Compositions and methods for rapid immunization against dengue virus

ABSTRACT

Embodiments of the present invention report compositions and methods for vaccinating a subject against all dengue virus serotypes. In some embodiments, multiple vaccine compositions may be administered to a subject in different anatomical locations in order to induce a rapid response to all dengue virus serotypes. In certain embodiments, administration of two or more vaccine compositions to a subject against all dengue virus serotypes may include two or more routes of administration.

PRIORITY

This application is a U.S. continuation application that claims priorityto U.S. patent application Ser. No. 14/407,012, filed Dec. 10, 2014, nowallowed, which is a national phase application filed under 35 U.S.C.§371 and claims priority to International Application No.PCT/US2013/045041, filed on Jun. 10, 2013, which is a continuationapplication of U.S. patent application Ser. No. 13/492,884 filed Jun.10, 2012, now U.S. Pat. No. 8,968,996, which is a continuation-in-partapplication and which claims the benefit under 35 U.S.C. §120 to U.S.application Ser. No. 12/790,511 filed May 28, 2010, now issued as U.S.Pat. No. 9,211,323, which claims priority under 35 U.S.C. §119(e) toU.S. Provisional Application Ser. No. 61/183,020, filed on Jun. 1, 2009.All prior applications are incorporated herein by reference in theirentirety for all purposes.

FEDERALLY FUNDED RESEARCH

This invention was made with Government support under U01 AI070443awarded by the National Institute of Health. The Government has certainrights in this invention.

FIELD

Embodiments of the present invention report compositions and methods foradministering a vaccine to a subject against all dengue virus strains.In some embodiments, vaccine compositions may be administered bysubcutaneous, intradermal, intramuscular or other injection orintroduction methods. In certain embodiments, injection in a subject ofa vaccine against all dengue virus types includes multiple anatomicalsites at day 0. Other embodiments include follow-on injections fromwithin days of the first treatment to up to 12 months after initialinjection(s). In other embodiments, no additional injections are neededother than the day 0 treatment. In certain embodiments, subcutaneous,intradermal, intramuscular or other modes of introducing to a subject, avaccine composition against dengue virus to provide protection againstthree or more of the dengue serotypes DEN-1, DEN-2, DEN-3 and DEN-4 uponadministration at day 0.

BACKGROUND

Vaccines for protection against viral infections have been effectivelyused to reduce the incidence of human disease. One of the mostsuccessful technologies for viral vaccines is to immunize animals orhumans with a weakened or attenuated strain of the virus (a “live,attenuated virus”). Due to limited replication after immunization, theattenuated strain does not cause disease. However, the limited viralreplication is sufficient to express the full repertoire of viralantigens and can generate potent and long-lasting immune responses tothe virus. Thus, upon subsequent exposure to a pathogenic strain of thevirus, the immunized individual is protected from disease. These live,attenuated viral vaccines are among the most successful vaccines used inpublic health.

SUMMARY

Embodiments of the present invention generally relate to methods andcompositions for inducing protection in a subject against multipledengue viruses by, for example, administering a multivalent denguevaccine to a subject. Some embodiments can include introducing a vaccinecomposition to a subject via intradermal (ID) injection. In accordancewith these embodiments, the vaccine composition can be introduced to asubject intradermally to, for example, to induce neutralizing antibodiesagainst three or more dengue virus serotypes. In certain embodiments, avaccine composition can include, but is not limited to, a single dose ofone formulation of a multivalent dengue serotype vaccine having apredetermined ratio administered to a subject. In other embodiments, avaccine composition may include, but is not limited to; an initial doseof one formulation of dengue vaccine (e.g. tetravalent formulations suchas DENVax™) and then one or more boosts of the same, or a differentformulation can be administered to a subject.

Other aspects herein can concern inducing a humoral or cellular immuneresponse in a subject by, for example, introducing a vaccine compositionto a subject via an intradermal route wherein the vaccine compositionincludes, but is not limited to, a dengue virus vaccine. In accordancewith these embodiments, compositions disclosed can be administeredintradermally to a subject for modulating neutralizing antibodyproduction in the subject against three or more dengue virus serotypes.Some aspects concern predetermined composition ratios (e.g. 1:1:1, 10:11:2:2, 1:10; 10:1, 3:4:3:3, 1:4:1; 5:5:4:5; or any ratio of three ormore serotypes is contemplated) of the various serotypes of dengue virusor fragments thereof or attenuated compositions thereof in a singlevaccine composition in order to increase cross protection and levels ofneutralizing antibodies in a subject against at least three dengue virusserotypes when the subject is administered the single vaccinecomposition.

In certain embodiments, some advantages of using intradermalintroduction of a vaccine against dengue virus can include, but are notlimited to, multiple protection (cross protection) against some or alldengue virus serotypes in a subject, reduced cost by using reducedvolumes of vaccine doses compared to subcutaneous injection, modulationof antibodies produced against some or all dengue virus serotypes in asubject and reduced pain at a site of administration in a subjectadministered a composition of vaccine against dengue virus.

In some embodiments, a single dose vaccine against dengue virus caninclude one or more dengue virus serotype(s). In addition, certainembodiments concern treating a subject with at least one additionalinjection(s) of a vaccine containing multiple dengue virusesadministered at a separate site from the first injection, for example,in close proximity to the initial injection or in a distant anatomicalsite on the subject. In addition, at least one additional intradermalinjection(s) may be performed less than 30 days after the firstadministration to the subject while others are performed 30 days and upto 12 months after the first administration of the vaccine.

Other embodiments disclosed herein relate to methods and compositionsfor inducing protection in a subject against all dengue virus serotypesby, for example, administering a vaccine to a subject against all denguevirus serotypes in two or more doses on one or more than one anatomicallocation consecutively within a short interval of time. Some embodimentscan include introducing a vaccine composition to a subject viaintradermal (ID), subcutaneous (SC), or intramuscular (IM) injection inone location and consecutively in another anatomical location by ID, SC,IM or by other introduction method at a second different anatomicallocation. Other embodiments include using any combination of modes ofadministration for introducing a dengue virus vaccine of all denguevirus serotypes to a subject where administration of the vaccine occursat two or more anatomical sites or by two or more different routesconsecutively on the same day to the subject.

Some embodiments include treating a subject in need of dengue virustetravalent vaccinations consecutively at two or more anatomicallocations. In certain embodiments, a subject may need two consecutiveadministrations in a single day to induce adequate levels ofneutralizing antibodies which will protect against dengue infection. Inother embodiments, a subject may be administered dengue virusmultivalent vaccinations consecutively at two or more anatomicallocations, then the subject can be administered at least a third vaccinewithin 30 days such as about 7, about 14, about 21 or about 28 dayslater with a composition comprising dengue virus serotypes which may ormay not have all serotypes. In other embodiments, a subject may beadministered dengue virus tetravalent vaccinations consecutively at twoor more anatomical locations on day 0, then the subject can beadministered at least a third vaccine within 30 days such as about 7,about 14, about 21 or about 28 days later with a composition comprisingdengue virus serotypes which may or may not have all serotypes. Vaccinecompositions of these and other embodiments disclosed herein may includetwo or more dengue virus serotypes at a predetermined ratio for thesubsequent administrations beyond the initial dual vaccination. Thesesubsequent vaccinations may depend on personalized titers of antibodiespost dual injection or other criteria such as results of testpopulations. In certain embodiments, a subsequent vaccination may onlyinclude a single dengue serotype (e.g. DEN-4).

In certain embodiments, the composition introduced to the subjectcomprises vaccines against all dengue virus serotypes, for exampletetravalent DENVax™ or another similar formulation. DENVax™ comprises atetravalent dengue vaccine of predetermined ratio where the vaccine ismade up of constructs on an attenuated DEN-2 backbone (see for example,PCT Application Number PCT/US01/05142 filed on Feb. 16, 2001incorporated herein by reference in its entirety for all purposes). Inother compositions, all dengue vaccine virus serotypes are in equalproportions in the composition. In yet other compositions, each denguevaccine virus serotype may be in a particular ratio to one another suchthat introduction of the composition induces sufficient levels ofneutralizing antibodies which would provide the subject with sufficientprotection against infection with three or more dengue viruses (e.g.DEN-1, DEN-2, DEN-3 and/or DEN-4). For example, if a subject, afterreceiving two or more compositions consecutively at two or moreanatomical locations and the subject has lower protection to one or moreparticular dengue virus serotypes, then a booster for that subject cancontain a multiple (more than two) vaccine components or a singlevaccine component to improve immune responses to all four dengue virusesin the subject. In accordance with these embodiments, samples from asubject may be analyzed for resistance to dengue infection usingstandard means known in the art.

In certain embodiments, the vaccine composition can be introduced to asubject by any route in multiple anatomical locations to, for example,protect against three or more dengue serotypes after consecutiveadministrations. In certain embodiments, a vaccine composition caninclude, but is not limited to, a single dose of a formulationcontaining all serotypes of dengue virus (e.g. DENVax™) administered toa subject capable of providing protection against at least three denguevirus serotypes. In other embodiments, a vaccine composition can includeattenuated dengue virus serotypes in combination with otheranti-pathogenic compositions (e.g. Japanese encephalitis, yellow fever,West Nile, influenza, Chikungunya or other). Compositions contemplatedherein can be administered by any method known in the art including, butnot limited to, intradermal, subcutaneous, intramuscular, intranasal,inhalation, vaginal, intravenous, ingested, and any other method.Introduction in two or more anatomical sites can include any combinationadministration including by the same mode in two or more anatomicalsites or by two or more different modes that include two or moreseparate anatomical sites. In accordance with these embodiments, two ormore anatomical sites can include different limbs. In other embodiments,vaccinations can be delivered to a subject using any device known in theart including, but not limited to, a needle and syringe, jet injection,microneedle injection, patch delivery (e.g. skin), intradermal deliverydevices, inhalation device, intranasal device, slow releasemicroparticles, and any other acceptable vaccine-delivery device.

In certain embodiments, a vaccine composition for dual administration ofdengue virus vaccines can include a composition comprising more than onechimeric dengue viruses in a single composition. In certaincompositions, the chimeric constructs used in such a composition aremade up of dengue-dengue serotypes such as a dengue-1, dengue-3, and/ordengue-4 on a dengue-2 backbone. In accordance with these embodiments, asingle vaccine composition can include live, attenuated dengue viruseswhere an immune response is induced in a subject receiving such acompositions to at least three and up to all four dengue virusserotypes. Constructs contemplated herein include live, attenuateddengue viruses comprising one or more live, attenuated dengue virusesand one or more dengue-dengue chimeric viruses further comprising capsidand non-structural proteins of the attenuated dengue virus andpre-membrane and envelope proteins of at least a second dengue virus ina single construct. In certain embodiments, the capsid andnon-structural proteins are from an attenuated dengue-1, dengue-2,dengue-3 or dengue-4 virus. In other embodiments, pre-membrane andenvelope proteins of at least a second dengue virus are dengue-2,dengue-3 or dengue-4 when the attenuated dengue virus is dengue-1; ordengue-1, dengue-3 or dengue-4 when the attenuated dengue virus isdengue-2; or dengue-1, dengue-2 or dengue-4 when the attenuated denguevirus is dengue-3; or dengue-1, dengue-2 or dengue-3 when the attenuateddengue virus is dengue-4. Further, dengue-dengue chimeric viruses caninclude the capsid and non-structural proteins of an attenuated dengue-2virus and the pre-membrane and envelope proteins are dengue-1, dengue-3or dengue-4.

Other embodiments include live, attenuated viruses where the backbone ofthe live attenuated virus is dengue-2. Further, dengue-2 can include anydengue-2 strain. In certain live attenuated dengue-2 viruses, dengue-2comprises PDK-53 strain. In another embodiment, a chimera is a nucleicacid chimera including a first nucleotide sequence encodingnonstructural proteins from an attenuated dengue-2 virus, and a secondnucleotide sequence encoding a structural protein from a secondflavivirus. In another embodiment, the structural protein can be the C,prM or E protein of a flavivirus. Examples of flaviviruses from whichthe structural protein may be selected include, but are not limited to,dengue-1 virus, dengue-2 virus, dengue-3 virus, dengue-4 virus, WestNile virus, Japanese encephalitis virus, St. Louis encephalitis virus,yellow fever virus and tick-borne encephalitis virus. In a furtherembodiment, the structural protein may be selected from non-flavivirusspecies that are closely related to the flaviviruses, such as hepatitisC virus.

In certain embodiments, amino acid substitution mutations in thenonstructural proteins and a nucleotide substitution mutation in the 5′noncoding region can be present. This nucleotide substitution mutationoccurs in the stem of a stem-loop structure that is conserved in allfour dengue serotypes. In particular, a single mutation at NS1-53, adouble mutation at NS1-53 and at 5′NC-57, a double mutation at NS1-53and at NS3-250, and a triple mutation at NS1-53, at 5′NC-57 and atNS3-250, can provide the attenuated DEN-2 virus disclosed herein.

It is contemplated that the genome of any dengue-2 virus containingnon-conservative amino acid substitutions at these loci can be used asthe backbone in the avirulent chimeras described herein. Furthermore,other flavivirus genomes containing analogous mutations at the sameloci, after amino acid sequence or nucleotide sequence alignment andstem structure analysis can also be used as the backbone structure andare defined herein as being equivalent to attenuating mutations of thedengue-2 PDK-53 genome. The backbone, that region of the chimera thatincludes 5′ and 3′ noncoding regions and the region encoding thenonstructural proteins, can also contain further mutations to maintainstability of the avirulent phenotype and to reduce the possibility thatthe avirulent virus or chimera might revert back to the virulentwild-type virus. For example, a second mutation in the stem of thestem/loop structure in the 5′ non-coding region can provide additionalstability, if desired.

In other embodiments, chimeric viruses can include nucleotide and aminoacid substitutions, deletions or insertions in their structural andnonstructural proteins in addition to those specifically describedherein. Structural and nonstructural proteins disclosed herein are to beunderstood to include any protein including or any gene encoding thesequence of the complete protein, an epitope of the protein, or anyfragment comprising, for example, two or more amino acid residuesthereof. Embodiments disclosed herein provide a method for makingchimeric viruses of embodiments described herein using recombinanttechniques, by inserting the required substitutions into the appropriatebackbone genome.

In other embodiments, compositions can include a pharmaceuticallyacceptable carrier and attenuated chimeric viruses which contain aminoacid sequences derived from other dengue virus serotypes, otherflavivirus species or other closely related species, such as hepatitis Cvirus. proteins or polypeptides comprising the amino acid sequencesderived from other dengue virus serotypes, other flavivirus species orother closely-related species, can act as immunogens and, thus, be usedto induce an immunogenic response against other dengue virus serotypes,other flavivirus species or other closely related species.

In one embodiment, nucleic acid chimeras including nucleotide sequencefrom an attenuated dengue-2 virus and nucleotide sequence from a seconddengue virus (or other flavivirus), wherein the nucleotide sequence fromthe second flavivirus directs the synthesis of flavivirus antigens arecontemplated of use for dual administration at day 0. In another aspectof the invention compositions for vaccines comprising three or moredengue virus serotypes is contemplated.

In another aspect, methods for making immunogenic or vaccinecompositions using recombinant techniques by inserting the requiredsubstitutions into an appropriate flavivirus genome. Another object ofthe invention is to provide compositions and methods for impartingimmunity against three or more dengue virus serotypes simultaneouslyusing dual administration in different anatomical areas to induce otherlymph nodes of a subject receiving such a regimen.

Another object of the invention is to provide nucleic acid probes andprimers for use in any of a number of rapid genetic tests that arediagnostic for each of the vaccine viruses of the current invention.This object of the invention may be embodied in polymerase chainreaction assays, hybridization assays or other nucleic acid sequencedetection techniques known to the art. One embodiment includes using anautomated PCR-based nucleic acid detection system.

In other embodiments, various mutations can be introduced to thechimeric dengue viruses in order to further attenuate the chimeric virusor improve immunogenicity. In certain embodiments, a composition caninclude chimeric dengue viruses capable of eliciting an immune responseto all four dengue virus serotypes wherein a single composition isintroduced in two anatomical locations of a subject. Certain embodimentsconcern targeting populations of people visiting dengue endemiccountries for short periods of time such as tourists.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments. Some embodimentsmay be better understood by reference to one or more of these drawingsalone or in combination with the detailed description of specificembodiments presented.

FIG. 1 represents an example of an intradermal injection devicecurrently available.

FIG. 2 represents examples of injection sites in a non-human primatesubject having intradermal introduction of a vaccine against denguevirus.

FIG. 3 represents a bar graph comparison of neutralizing antibody titerproduced against different ratios of dengue virus serotypes after a one(primary) administration via the subcutaneous (SC) versus intradermal(ID) route of injection of a vaccine against dengue virus.

FIG. 4 represents a bar graph comparison of neutralizing antibody titerproduced against different dengue virus serotypes after a second,boosting administration via the subcutaneous (SC) versus intradermal(ID) injection of a vaccine against dengue virus.

FIG. 5 represents a histogram plot of neutralizing antibody titers aftersubcutaneous and intradermal immunizations with a vaccine against adengue virus serotype-4 in mice.

FIGS. 6A and 6B represent graphic depictions of mouse survival aftervaccination with a dengue vaccine followed by a challenge with wild-typedengue virus. Mice were vaccinated by SC or ID route of infection with adengue vaccine (e.g. DENVax-4) or a buffer/placebo (e.g. TFA).

FIGS. 7A-7D represent neutralizing antibody titers for DEN-1, DEN-2,DEN-3 and DEN-4 at day 28 and day 56 after two day-0; or 1 day-0 and 1day-42 injections (e.g. DENVax™; 4:3:4:5 ratio).

FIGS. 8A-8D represent neutralizing antibody titers for DEN-1, DEN-2,DEN-3 and DEN-4 at day 28 and day 56 after two day-0; or 1 day-0 and 1day-42 injections (e.g. DENVax™; 3:3:3:3, approximately equivalentamounts used).

FIGS. 9A-9D represent graphs comparing neutralizing antibody titersachieved in non-human primates after SC immunization with tetravalentdengue virus vaccines. Two groups were vaccinated with the needle-freedevice via the subcutaneous route either twice on the same day (0,0) oronce on day 0 and again on day 60 (0,60).

FIGS. 10A-10B represent data obtained from a human clinical trial.Seronegative humans (humans demonstrating little to no antibodies todengue virus serotypes at the onset of the trial) were given two dosesof a tetravalent serotype formulation of dengue vaccine eithersubcutaneously or intradermally (day 0 and day 90). Antibody levelsagainst each of the dengue serotypes were analyzed on days 0, 30, 60, 90and 120.

FIGS. 11A-11D represent a graph comparing neutralizing antibody titersachieved in non-human primates after subcutaneous immunization with atetravalent serotype dengue vaccine. Two groups were vaccinated eithertwice on the same day (0,0) or once on day 0 and again on day 60 (0,60).Serum was analyzed for presence of antibodies on days 0, 28, 58, 73 and90, and the detection of antibodies against all four dengue serotypeswere analyzed (DEN-1, DEN-2, DEN-3, DEN-4).

FIG. 12 represents gene expression levels in samples obtained from asubject having single administration or dual (double) administration atseparate anatomical sites of dengue virus vaccines. This data representsgene cluster variability where changes in levels of various genetranscripts in a subject are analyzed after exposure to a compositionusing certain regimens disclosed herein.

FIGS. 13A-13F represent levels of certain genes after single or dual(double) administration. The genes represented in cluster 2 FIG. 12 andA-F are associated with innate immunity.

DEFINITIONS

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, vessel can include, but is not limited to, test tube,mini- or micro-fuge tube, channel, vial, microtiter plate or container.

As used herein the specification, “subject” or “subjects” may includebut are not limited mammals such as humans or mammals, domesticated orwild, for example dogs, cats, other household pets (e.g., hamster,guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairiedogs, or zoo animals.

As used herein, “about” or “approximately” can mean plus or minus tenpercent.

As used herein, “attenuated virus” can mean a virus that demonstratesreduced or no clinical signs of disease when administered to a subjectsuch as a mammal (e.g., human or an animal).

As used herein, “consecutively” can mean in close temporal proximity,usually within a single patient visit and within 24 hours.

As used herein, “administration” can mean delivery of a vaccine ortherapy to an individual animal or human by any one of many methods suchas intradermal, subcutaneous, intramuscular, intranasal, inhalation,vaginal, intravenous, oral, buccal, by inhalation, intranasally, or anyothers known in the art.

DESCRIPTION

In the following sections, various exemplary compositions and methodsare described in order to detail various embodiments. It will be obviousto one skilled in the art that practicing the various embodiments doesnot require the employment of all or even some of the details outlinedherein, but rather that concentrations, times and other details may bemodified through routine experimentation. In some cases, well-knownmethods or components have not been included in the description.

Certain aspects of the present invention include, but are not limitedto, administration of vaccine compositions against dengue virus.

Embodiments of the present invention generally relate to methods andcompositions for inducing protective neutralizing antibodies in asubject against three or more dengue virus serotypes. Other embodimentscan include introducing a vaccine composition to a subject via anymethod known in the art including, but not limited to, intradermal,subcutaneous, intramuscular, intranasal, inhalation, orally,intranasally, vaginal, intravenous, ingested, and any other methodwherein the vaccine composition so introduced induces neutralizingantibodies against three or more dengue virus serotypes. In certainembodiments, the vaccine composition comprises a dose of a vaccineagainst three or more dengue virus serotypes administered to a subject.In other embodiments, the vaccine composition comprises an initial doseagainst all four dengue serotypes then, one or more other vaccinecompositions administered to a subject.

Other aspects of the present invention include modulating an immuneresponse to a vaccine against dengue virus administered intradermallycompared to subcutaneously to a subject. Vaccines against dengue virusmay include a composition comprising predetermined ratios of all fourlive, attenuated dengue vaccine viruses, recombinant dengue vaccineviruses, chimeric viruses or mutants thereof. The ratios of variousdengue serotypes may be equivalent or nearly equal in representation orcertain serotypes may be represented at higher concentrations thanothers depending on need or ability to induce a balanced neutralizingantibody response in the subject. In accordance with these embodiments,ratios of different dengue vaccines may differ by 2 to 100,000 fold(e.g. plaque forming units) between any two serotypes. This can dependon, for example, number of serotypes represented in the formulation,predetermined response and desired effect. It is contemplated that anydengue vaccine virus serotype formulation may be used to generate avaccine (e.g. attenuated virus etc.) of use in consecutiveadministration to a subject in need thereof where the compositionincludes, but is not limited to, three or more dengue virus serotypes.

In other embodiments, compositions of dengue virus vaccine formulationsmay be introduced to a subject prior to, during or after exposure todengue virus by the subject. In accordance with these embodiments, asubject may receive more than one administration consecutively or morethan one administration comprising a dengue virus formulation,optionally, followed by one or more additional administrations at alater time. Intradermal, subcutaneous, intramuscular, intranasal,inhalation, vaginal, intravenous, oral, and any other method ofapplications of formulations described herein may be combined with anyother anti-viral treatment. In some embodiments, it is contemplated thatintradermal, subcutaneous, intramuscular introduction of a formulationcontemplated herein may be administered to any appropriate region of asubject's body (e.g. arm, shoulder, hip, intranasally etc). In addition,parenteral administration of vaccine formulations may be combined withother modes of administration such as intranasal, pulmonary, oral,buccal, or vaginal in consecutive administrations. In some embodiments,it is contemplated that, after consecutive administrations as describedherein primary or booster administrations may occur consecutively on thesame day, consecutive days, weekly, monthly, bi-monthly or otherappropriate treatment regimen.

Dengue is endemic in Asia, Central and South America including Colombia,the Caribbean, the Pacific Islands, and parts of Africa and Australia.It is estimated that 3.6 billion people (55% of the world's population)live in areas at risk of dengue virus transmission (DVI). Infection witha dengue virus can result in a range of symptoms, from subclinicaldisease to debilitating but transient dengue fever to life-threateningdengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS).Currently, there is no therapeutic treatment or prophylactic vaccine fordengue fever. Given the impact of dengue on populations in endemiccountries and on travelers to those regions, a vaccine to prevent dengueis needed.

Dengue is a mosquito borne viral disease, transmitted from human tohuman primarily by the mosquito, Aedes aegypti. Dengue viruses (DEN)contain a single-stranded, positive-sense RNA genome of approximately 11kb. The genome consists of three structural proteins, capsid (C),premembrane (prM), and envelope (E), and seven nonstructural proteins,NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NSS. There are four differentserotypes of dengue viruses, DEN-1, DEN-2, DEN-3 and DEN-4. Primaryinfection with a given serotype induces lifelong serotype specificimmunity. However, there is no long-term cross-protective immunityagainst the other three dengue virus serotypes, and subsequent infectionwith an alternate serotype leads to increased probability of more severedisease, such as DHF or DSS.

Due to the disease enhancement associated with secondary DENYinfections, a multivalent (e.g. tetravalent) vaccine that stimulatesimmunity against more than one and up to all four serotypes of DENY isneeded. Several DENY vaccine candidates attenuated by classical serialpassage in cell culture have proven unsafe or poorly immunogenic.Chimeric live-attenuated, recombinant DENY vaccines candidates,including viruses based on the attenuated genetic background of yellowfever 17D (YF-17D) vaccine virus, DENY-2 PDK-53 vaccine virus, or DENY-4containing a 30-nucleotide 3′ non-coding region (NCR) deletion are knownin the art.

A challenging issue in the development of an effective live-attenuateddengue virus (DENY) vaccine is the interference between the four denguevaccine viruses when administered as a tetravalent formulation.Interference is manifest when one or more components of a multivalentmixture will induce lower immune responses than those elicited by eachindividual monovalent vaccine. Interference has been observed withvaccines for diseases with multiple pathogenic serotypes, such as polio,dengue or others. Due in part to this interference, it was previouslydiscovered that three dose regimen of oral polio vaccine is required toinduce adequate immune responses to the three key serotypes.Historically studies with live attenuated tetravalent dengue vaccineshave shown that the DENY serotype that elicits the strongestneutralizing antibody response when administered alone tends to dominateimmune responses when administered in the context of a multivalentformulation containing other serotypes. As an example, tetravalentmixtures of four different live, attenuated dengue vaccines showeddominant responses to the DEN-3 component and reduced immune responsesto DEN-1, -2 and -4 (see for example, Sabchareon, et al., 2002,Kitchener, et al. 2006). As a result of this dominance, clinicaldevelopment of the tetravalent mixtures was suspended. Interference hasbeen seen with recombinant, live attenuated viruses as well.Interference was documented in tetravalent mixtures of dengue/yellowfever chimeras (Guy, et al. 2009. Evaluation of Interferences betweenDengue Vaccine Serotypes in a Monkey Model. Am. J. Trop Med. Hyg. 80:3012-311). In these studies, two serotypes were found to dominate theresponses in tetravalent formulations of ChimeriVax vaccine strains.Interference could be overcome by administering two bivalent vaccineformulations, either in separate anatomical locations or sequentially intime, or by a third administration of the tetravalent formulation afterone year. Similarly, it was demonstrated that improved multivalentresponses with tetravalent recombinant vaccine strains (in this case,formulations containing DENV or chimeric DENV with deletions in the 3′non-coding region) could be obtained only with a prolonged four monthinternal between the first and second administration. (Blaney, et al.,2005. Recombinant, Live-Attenuated Tetravalent Dengue Virus VaccineFormulations Induce a Balanced, Broad, and Protective NeutralizingAntibody Response against Each of the Four Serotypes in Rhesus Monkeys.J. Virology 79: 5516-5528).

Successful vaccination often requires vaccine delivery to closely mimicnatural infection. To date, all clinical trials of dengue candidatevaccines have utilized the SC route using needle and syringe. Thenatural route of dengue infection is through mosquito transmission inthe dermis. The skin is thought to be an immuno-competent organfunctioning as an immune barrier to infections A highly dense network ofspecialized antigen-presenting cells (APCs, such as Langerhan's cellsand dendritic cells) are present in the epidermis and serve to protectthe host against infectious pathogens through efficient uptake andpresentation of antigens to the regional lymph nodes. Both these subsetsof APCs together with resident macrophages have been shown to be naturaltargets of dengue virus infection. Given the fact that the epidermis isrich in immunocompetent cells, it was contemplated herein that the useof intradermal route for dengue virus vaccine delivery will favor theinduction of more potent and balanced immune responses to all fourdengue virus serotypes. Specifically, the presence of an increasednumber of natural host cells in the skin for virus replication mayreduce interference and permit replication of the less dominant virusesin tetravalent formulations. In certain embodiments, intradermalimmunization of multivalent, live, attenuated dengue vaccines can beused to induce more balanced immune responses to dengue virus exposurein a subject.

Certain embodiments disclosed herein concern DENVax™. DENVax™ is adengue vaccine that consists of a mixture of four recombinant denguevirus strains designed to generate immune responses to the four dengueserotypes (DEN-1, DEN-2, DEN-3 and DEN-4). Not to be bound by anylimitations to a particular tetravalent formulation, DENVax™, the dengueserotype 2 vaccine component (DENVax-2) corresponds to an attenuatedDEN-2 PDK-53 strain. This construct has already been investigated inmany clinical studies. The other dengue vaccine strains (DENVax-1,DENVax-3 and DENVax-4) are chimeras consisting of the DEN-1, DEN-3 orDEN-4 structural pre-membrane (prM) and envelope (E) protein genescloned into a DEN-2 PDK-53 non-structural gene backbone. Theserecombinant viruses express the surface antigens of DEN-1, DEN-3 orDEN-4 and retain the genetic alterations responsible for the attenuationof the DEN-2 PDK-53 strain. In certain embodiments, DENVax™ can be usedas an example of a multivalent live, attenuated dengue vaccine havingall four dengue virus serotypes represented in one vaccine compositionat various ratios. Other embodiments relate to optimizing tetravalentvaccine administrations. Yet other embodiments relate to DENVax™immunization methods.

During the course of exploring intradermal delivery of multivalentdengue vaccines, it was discovered that administration of more than onedose of a multivalent vaccine in at least two separate anatomical sitesinduced neutralizing antibody responses that were approximatelyequivalent or superior to administering multiple doses separated bytime. Further, it was discovered that the benefit of multiple siteadministration was independent of the route of immunization.

This finding was unexpected. Information was previously disclosedregarding multiple subcutaneous administrations of a tetravalent vaccinebased on deleted, attenuated and/or recombinant viruses. It was reportedthat a second administration of a tetravalent administration 30 daysafter the first administration failed to increase neutralizing antibodytiters. In contrast, a second administration 120 days after the first,improved neutralizing antibody titers to all four dengue serotypes.Similar information was reported in clinical trials of yellowfever/dengue recombinant vaccines (Poo, et al. 2011 Ped. Inf. Dis J. 30:1-9) It was also suggested that a three month interval betweenadministrations was suboptimal for generating neutralizing antibodyresponse against multiple dengue viruses. (Capeding et al. 2011 Vaccine29: 3863-3872) These reports regarding two clinical studies suggestedthat longer intervals such as 6-9 months are required to generate bettermultivalent immune responses. Lastly, in early human challenge studies,it was reported that wild type dengue viruses elicit broadlycross-reactive antibodies that persist for up to 6 months after initialinfection. These data support the concept that a short immunizationregimen are suboptimal for live attenuated vaccines: the transientcross-reactive antibodies previously observed would effectivelyneutralize any of the live, attenuated vaccine components in amultivalent formulation. Until the instant disclosure, immunizationregimens with multivalent, live attenuated vaccines at shorter intervalsin more than one anatomical site were not considered a viable option fortreating a subject in need of such a treatment. It is contemplatedherein that multiple site administration, by accessing larger numbers ofantigen presenting cells and/or more than one draining lymph node,permits immune responses to less dominant components of a multivalent,live attenuated vaccine and effectively reduces vaccine interference.

In certain embodiments, the composition introduced to the subjectcomprises vaccines against all dengue virus serotypes (DEN-1, DEN-2,DEN-3, DEN-4). In other embodiments, a composition contemplated hereincan include DENVax™ or other similar formulation. In some compositions,vaccine viruses against all dengue serotypes are in equal proportions inthe composition. In yet other compositions, each dengue vaccine virusserotype may be in a particular ratio to one another such thatintroduction of the composition provides the subject with sufficientlevels of neutralizing antibodies against all dengue viruses (e.g.DEN-1, DEN-2, DEN-3, DEN-4).

In certain embodiments, a vaccine composition for dual administration ofdengue virus vaccines can include a composition comprising more than onechimeric dengue viruses in a single composition. In certaincompositions, the chimeric constructs used in such a composition aremade up of dengue-dengue serotypes such as a dengue-1, dengue-3, and/ordengue-4 on a dengue-2 backbone. In accordance with these embodiments, asingle vaccine composition can include live, attenuated dengue viruseswhere an immune response is induced in a subject receiving such acompositions to at least three and up to all four dengue virusserotypes. Constructs contemplated herein include live, attenuateddengue viruses comprising one or more live, attenuated dengue virusesand one or more dengue-dengue chimeric viruses further comprising capsidand non-structural proteins of the attenuated dengue virus andpre-membrane and envelope proteins of at least a second dengue virus ina single construct. In certain embodiments, the capsid andnon-structural proteins are from an attenuated dengue-1, dengue-2,dengue-3 or dengue-4 virus. In other embodiments, pre-membrane andenvelope proteins of at least a second dengue virus are dengue-2,dengue-3 or dengue-4 when the attenuated dengue virus is dengue-1; ordengue-1, dengue-3 or dengue-4 when the attenuated dengue virus isdengue-2; or dengue-1, dengue-2 or dengue-4 when the attenuated denguevirus is dengue-3; or dengue-1, dengue-2 or dengue-3 when the attenuateddengue virus is dengue-4. Further, dengue-dengue chimeric viruses caninclude the capsid and non-structural proteins of an attenuated dengue-2virus and the pre-membrane and envelope proteins are dengue-1, dengue-3or dengue-4.

Other embodiments include live, attenuated viruses where the backbone ofthe live attenuated virus is dengue-2. Further, dengue-2 can include anydengue-2 strain. In certain live attenuated dengue-2 viruses, dengue-2comprises PDK-53 strain. In another embodiment, a chimera is a nucleicacid chimera including a first nucleotide sequence encodingnonstructural proteins from an attenuated dengue-2 virus, and a secondnucleotide sequence encoding a structural protein from a secondflavivirus. In another embodiment, the structural protein can be the C,prM or E protein of a flavivirus. Examples of flaviviruses from whichthe structural protein may be selected include, but are not limited to,dengue-1 virus, dengue-2 virus, dengue-3 virus, dengue-4 virus, WestNile virus, Japanese encephalitis virus, St. Louis encephalitis virus,yellow fever virus and tick-borne encephalitis virus. In a furtherembodiment, the structural protein may be selected from non-flavivirusspecies that are closely related to the flaviviruses, such as hepatitisC virus.

In certain embodiments, amino acid substitution mutations in thenonstructural proteins and a nucleotide substitution mutation in the 5′noncoding region can be present. This nucleotide substitution mutationoccurs in the stem of a stem-loop structure that is conserved in allfour dengue serotypes. In particular, a single mutation at NS1-53, adouble mutation at NS1-53 and at 5′NC-57, a double mutation at NS1-53and at NS3-250, and a triple mutation at NS1-53, at 5′NC-57 and atNS3-250, can provide the attenuated DEN-2 virus disclosed herein

It is contemplated that the genome of any dengue-2 virus containingnon-conservative amino acid substitutions at these loci can be used asthe backbone in the avirulent chimeras described herein. Furthermore,other flavivirus genomes containing analogous mutations at the sameloci, after amino acid sequence or nucleotide sequence alignment andstem structure analysis can also be used as the backbone structure andare defined herein as being equivalent to attenuating mutations of thedengue-2 PDK-53 genome. The backbone, that region of the chimera thatincludes 5′ and 3′ noncoding regions and the region encoding thenonstructural proteins, can also contain further mutations to maintainstability of the avirulent phenotype and to reduce the possibility thatthe avirulent virus or chimera might revert back to the virulentwild-type virus. For example, a second mutation in the stem of thestem/loop structure in the 5′ non-coding region can provide additionalstability, if desired.

In other embodiments, chimeric viruses can include nucleotide and aminoacid substitutions, deletions or insertions in their structural andnonstructural proteins in addition to those specifically describedherein. Structural and nonstructural proteins disclosed herein are to beunderstood to include any protein including or any gene encoding thesequence of the complete protein, an epitope of the protein, or anyfragment comprising, for example, two or more amino acid residuesthereof. Embodiments disclosed herein provide a method for makingchimeric viruses of embodiments described herein using recombinanttechniques, by inserting the required substitutions into the appropriatebackbone genome.

In other embodiments, compositions can include a pharmaceuticallyacceptable carrier and attenuated chimeric viruses which contain aminoacid sequences derived from other dengue virus serotypes, otherflavivirus species or other closely related species, such as hepatitis Cvirus. proteins or polypeptides comprising the amino acid sequencesderived from other dengue virus serotypes, other flavivirus species orother closely-related species, can act as immunogens and, thus, be usedto induce an immunogenic response against other dengue virus serotypes,other flavivirus species or other closely related species.

In one embodiment, nucleic acid chimeras including nucleotide sequencefrom an attenuated dengue-2 virus and nucleotide sequence from a seconddengue virus (or other flavivirus), wherein the nucleotide sequence fromthe second flavivirus directs the synthesis of flavivirus antigens arecontemplated of use for dual administration at day 0. In another aspectof the invention compositions for vaccines comprising three or moredengue virus serotypes is contemplated.

In another aspect, methods for making immunogenic or vaccinecompositions using recombinant techniques by inserting the requiredsubstitutions into an appropriate flavivirus genome. Another object ofthe invention is to provide compositions and methods for impartingimmunity against three or more dengue virus serotypes simultaneouslyusing dual administration in different anatomical areas to induce otherlymph nodes of a subject receiving such a regimen.

Another object of the invention is to provide nucleic acid probes andprimers for use in any of a number of rapid genetic tests that arediagnostic for each of the vaccine viruses of the current invention.This object of the invention may be embodied in polymerase chainreaction assays, hybridization assays or other nucleic acid sequencedetection techniques known to the art. One embodiment includes using anautomated PCR-based nucleic acid detection system.

In other embodiments, various mutations can be introduced to thechimeric dengue viruses in order to further attenuate the chimeric virusor improve immunogenicity. In certain embodiments, a composition caninclude chimeric dengue viruses capable of eliciting an immune responseto all four dengue virus serotypes wherein a single composition isintroduced in two anatomical locations of a subject. Certain embodimentsconcern targeting populations of people visiting dengue endemiccountries for short periods of time such as tourists.

Certain embodiments disclosed herein relate to methods and compositionsfor a rapid induction of protection in a subject against all denguevirus serotypes by, for example, administering a vaccine to a subjectagainst all dengue virus serotypes in more than one anatomical locationconsecutively on the same day. Some embodiments can include introducinga vaccine composition to a subject via intradermal (ID) or subcutaneous(SC) injection or other administration mode in one anatomical locationthen introducing at least a second vaccine composition at anotheranatomical location by ID, SC or other administration mode. Someembodiments include using any combination of modes of administration forintroducing a dengue virus vaccine of all dengue virus serotypes to asubject where administration of the vaccine occurs at two or moreanatomical sites or by two or more different routes on day 0 to thesubject. Some embodiments include using the same mode of administrationbut at different anatomical locations.

Some dengue virus vaccine compositions described herein range in dosagefrom from 10² to 5×10⁶ PFU for each serotype in a composition. Othercompositions (e.g. follow-on vaccinations) contemplated herein includecompositions that have dosages less than or more than this range basedon immune response in the subject after primary immunization. In certainembodiments, ratios can vary for the various Dengue vaccine virusserotypes depending on need and immune response in a subject.

In certain embodiments, compositions introduced on the first vaccinationor in any follow-on vaccination contemplated herein may include onetetravalent dengue virus composition. In accordance with theseembodiments, the composition can include DENVax™ or other similartetravalent formulation of equal or equivalent ratios or atpredetermined serotype ratios. Other embodiments, can include usingdifferent formulations (e.g. serotype ratios) for each of the vaccinecompositions administered at the primary vaccination or any follow-onvaccinations (e.g. less than 30 days later).

Some embodiments herein include treating a subject in need of such avaccine, on day 0 at two or more anatomical locations then administeringat least a second vaccine within 30 days such as about 7, about 14,about 21 or about 28 days later with a composition comprising denguevirus serotypes which may or may not have all serotypes. In certainembodiments, each vaccination has all dengue virus serotypes representedin the vaccine formulation. Vaccine compositions of follow-onadministration disclosed herein may include two or more dengue virusserotypes at a predetermined ratio for the subsequent administration(s).

In certain embodiments, the composition introduced to the subjectcomprises all dengue virus serotypes. In some embodiments, vaccinecompositions comprise various formulations of DENVax™ or other similarformulation. In certain vaccine compositions, the ratio ofDEN-1:DEN-2:DEN-3:DEN-4 can be 3:3:3:3, 4:3:4:5, 5:4:5:5, 5:4:5:5,5:5:5:5, 5:5:5; 10, 10:1:10:100 or other ratio where the ratio between 2serotypes can be about 2 to about 100,000 fold difference (e.g.DENVax_(4:3:4:5)™ etc.) in a single composition. In certain embodimentsa dengue serotype ratio can be DEN-1 at 2×10⁴: DEN-2 at 5×10⁴: DEN-3 at1×10⁵: DEN-4 at 3×10⁵ PFUs or DEN-1 at 8×10³: DEN-2 at 5×10³: DEN-3 at1×10⁴: DEN-4 at 2×10⁵ PFUs. In some compositions, all dengue vaccinevirus serotypes are in equal proportions in the composition. In yetother compositions, each dengue vaccine virus serotype may be in aparticular ratio to another serotype such that introduction of thecomposition provides the subject with adequate or more than adequatelevels of neutralizing antibodies which confer protection against alldengue viruses (e.g. Dengue 1, 2, 3 and 4). For example, if afterreceiving two or more consecutive vaccinations on day 0 at two or moreanatomical locations, the subject has lower protection to one or moreparticular dengue virus serotypes, then a booster for that subject cancontain an increased concentration of the one or more dengue vaccinevirus serotype (that demonstrated lower neutralizing antibodies) toprovide better protection against all dengue virus types. In accordancewith these embodiments, samples from a subject may be analyzed for animmune response to dengue serotype infection (e.g. Dengue-1, -2, -3, -4)using standard means known in the art.

In certain embodiments, the vaccine composition can be simultaneously orconsecutively introduced to a subject intradermally in multipleanatomical locations to, for example, protect against all dengueserotypes (e.g. cross protection). In certain embodiments, a vaccinecomposition can include, but is not limited to, a single formulation ofall dengue vaccine virus serotypes (e.g. DENVax™) administered to asubject capable of providing full protection against infection by alldengue virus serotypes. In other embodiments, a vaccine composition caninclude attenuated dengue virus serotypes in combination with otheranti-pathogenic compositions (e.g. Japanese encephalitis, West Nile,influenza etc.). Compositions contemplated herein can be administered byany method known in the art including, but not limited to, intradermal,subcutaneous, intramuscular, intranasal, inhalation, vaginal,intravenous, ingested, and any other method. Introduction in two or moreanatomical sites can include any combination administration including bythe same mode in two or more anatomical sites or by two different modesthat include two separate anatomical sites. In accordance with theseembodiments, two or more anatomical sites can include different limbs.

For example, if a subject, after receiving two or more consecutivevaccinations on day 0 at two or more anatomical locations and thesubject does not induce poor levels of neutralizing antibodies to one ormore particular dengue virus serotypes, then a booster vaccination forthat subject can contain an increased concentration of the one or moredengue vaccine virus serotype (that demonstrated lower levels ofneutralizing antibodies) to provide complete protection againstinfection by all dengue virus types. In accordance with theseembodiments, samples from a subject may be analyzed for resistance todengue infection using standard means known in the art.

In certain embodiments, doses of the vaccine composition can beconsecutively introduced to a subject in multiple anatomical locationsto, for example, to protect against all dengue serotypes (e.g. crossprotection) at day 0. In certain embodiments, a vaccine composition caninclude, but is not limited to, a single composition of three or fourdengue virus serotypes (e.g. DENVax™) administered to a subject capableof inducing neutralizing antibodies to levels which would provide fullprotection against infection by all dengue virus serotypes. Thus, aparticular subject may need to visit a clinic only one time to receiveenough protection to visit or remain in a region having dengue virus fora predetermined period of time (e.g. 30 days). In other embodiments, avaccine composition can include attenuated dengue virus serotypes incombination with vaccine compositions against other pathogens (e.g.flaviviruses such as Japanese encephalitis, West Nile, or other virusessuch as influenza etc.). Compositions contemplated herein can beadministered by any method known in the art including, but not limitedto, intradermal, subcutaneous, intramuscular, intranasal, inhalation,vaginal, intravenous, ingested, and any other method. Introduction intwo or more anatomical sites can include any combination administrationincluding by the same mode in two or more anatomical sites or by two ormore different modes that include two or more separate anatomical sites.In accordance with these embodiments, two or more anatomical sites caninclude different limbs, different tissues, intranasally, as drops (e.g.for the eye), intramuscular in two or more locations.

In certain embodiments, vaccine compositions disclosed herein can bechimeric constructs that can include a mixture of constructs that makeup at least 3 dengue serotypes in a vaccine composition foradministration to a subject. In other embodiments, dengue virus vaccinescan include constructs having an attenuated flavivirus backbone withvarious dengue serotype substitutions representing each of the fourserotypes where the constructs can be mixed in a composition foradministration as a vaccine.

Chimeras contemplated and described herein can be produced by splicingone or more of the structural protein genes of the flavivirus againstwhich immunity is desired into a a dengue virus genome backbone (e.g.PDK-53), or the equivalent thereof as described above, using recombinantengineering techniques well known to those skilled in the art to removethe corresponding structural genes and replace it with the desiredstructural gene. Alternatively, using the sequences provided in thesequence listing, the nucleic acid molecules encoding the flavivirusproteins may be synthesized using known nucleic acid synthesistechniques and inserted into an appropriate vector. Avirulent,immunogenic virus is therefore produced using recombinant engineeringtechniques known to those skilled in the art.

As mentioned above, the gene to be inserted into the backbone encodes aflavivirus (e.g. other dengue virus serotype) structural protein.Preferably the flavivirus gene to be inserted is a gene encoding a Cprotein, a PrM protein and/or an E protein. The sequence inserted intothe dengue-2 backbone can encode both the PrM and E structural proteins.The sequence inserted into the dengue-2 backbone can encode the C, prMand E structural proteins. The dengue virus backbone is the PDK-53dengue-2 virus genome and includes either the spliced genes that encodethe C, PrM and/or E structural proteins of dengue-1 (DEN-2/1), thespliced genes that encode the PrM and/or E structural proteins ofdengue-3 (DEN-2/3), or the spliced genes encode the PrM and/or Estructural proteins of dengue-4 (DEN-2/4). In one embodiment, thespliced gene that encodes the structural protein of dengue-3 virusdirects the synthesis of an E protein that contains a leucine at aminoacid position 345.

In another embodiment, a chimera of encodes the C structural protein ofdengue-2 virus and directs the synthesis of a C protein that contains aserine at amino acid position 100 and comprises a spliced gene encodingthe structural proteins of dengue-4 which directs the synthesis of an Eprotein that contains a leucine at amino acid position 447.

In yet other embodiments, a chimera can encode the C structural proteinof dengue-2 virus and directs the synthesis of a C protein that containsa serine at amino acid position 100 and comprises a spliced geneencoding the structural proteins of dengue-4 which directs the synthesisof an E protein that contains a leucine at amino acid position 447 and avaline at amino acid position 364. The structural proteins describedherein can be present as the only flavivirus structural protein or inany combination of flavivirus structural proteins in a viral chimera ofthis invention.

Chimeras can be engineered by recombination of full genome-length cDNAclones derived from both DEN-2 16681 wild type virus and either of thePDK-53 dengue-2 virus variants. Uncloned PDK-53 vaccine contains amixture of two genotypic variants, designated herein as PDK53-E andPDK53-V. The PDK53-V variant contains all nine PDK-53 vaccine-specificnucleotide mutations, including the Glu-to-Val mutation at amino acidposition NS3-250. The PDK53-E variant contains eight of the ninemutations of the PDK-53 vaccine and the NS3-250-Glu of the parental16681 virus. Infectious cDNA clones are constructed for both variants,and viruses derived from both clones are attenuated in mice. Thephenotypic markers of attenuation of DEN-2 PDK-53 virus include smallplaque size, temperature sensitivity (particularly in LLC-MK.sub.2cells), limited replication (particularly in C6/36 cells), attenuationfor newborn mice (specifically loss of neurovirulence for suckling mice)and decreased incidence of viremia in monkeys. The chimeras that areuseful as vaccine candidates are constructed in the genetic backgroundsof the two DEN-2 PDK-53 variants which all contain mutations innonstructural regions of the genome, including 5′NC-57 C-to-T(16681-to-PDK-53) in the 5′ noncoding region, as well as mutations inthe amino acid sequence of the nonstructural proteins, such as, forexample, NS1-53 Gly-to-Asp and NS3-250 Glu-to-Val.

Suitable chimeric viruses or nucleic acid chimeras containing nucleotidesequences encoding structural proteins of other flaviviruses or denguevirus serotypes can be evaluated for usefulness as vaccines by screeningthem for the foregoing phenotypic markers of attenuation that indicateavirulence and by screening them for immunogenicity. Antigenicity andimmunogenicity can be evaluated using in vitro or in vivo reactivitywith flavivirus antibodies or immunoreactive serum using routinescreening procedures known to those skilled in the art.

Flavivirus Vaccines

In certain embodiments, chimeric viruses and nucleic acid chimerasprovide live, attenuated viruses useful as immunogens or vaccines. Thesechimeras exhibit high immunogenicity while at the same time producing nodangerous pathogenic or lethal effects.

Effective vaccination against all strains of dengue virus has beendifficult. To prevent the possible occurrence of DHF/DSS in patientsvaccinated against only one serotype of dengue virus, rapid immunizationusing a trivalent or tetravalent dengue virus vaccine is needed toprovide simultaneous immunity for all four serotypes of the virus. Onetetravalent vaccine is produced by combining dengue-2 PDK-53 with thedengue-2/1, dengue-2/3, and dengue-2/4 chimeras described above in asuitable pharmaceutical carrier for administration as a multivalentvaccine.

The chimeric viruses or nucleic acid chimeras can include structuralgenes of either wild-type or attenuated virus in a virulent or anattenuated DEN-2 virus backbone. For example, the chimera may expressthe structural protein genes of wild-type DEN-1 16007 virus or itscandidate PDK-13 vaccine derivative in either of the DEN-2 PDK-53backgrounds.

Tetravalent formulations, e.g. DENVax™, can be prepared by mixingpredetermined amounts of each monovalent vaccine component or chimericconstructs. Based on input titer of each vaccine component, a definedvolume of monovalent vaccines can be added to a final volume of either0.1 mL (e.g. for intradermal) or 0.5 mL (e.g. for subcutaneous) vaccineformulation. The remaining volume of the tetravalent DENVax™ vaccine canbe composed of diluent containing Trehalose (15%) F127 (1%) and humanserum albumin (0.1%) in a saline buffer to stabilize the live,attenuated vaccine formulation. In certain embodiments, a predeterminedratio of at least three dengue virus serotypes can be represented in asingle composition. For example, dengue-1 thru dengue-4 constructs maybe represented in a single composition where more of one serotype of alive, attenuated virus can be present compared to the other constructs.For example, dengue-4 can be several fold pfu higher than other dengueviruses because it can demonstrate a reduced response.

Methods Nucleic Acid Amplification

Nucleic acids may be used in any formulation or used to generate anyformulation contemplated herein. Nucleic acid sequences used as atemplate for amplification can be isolated viruses (e.g. dengueviruses), according to standard methodologies. A nucleic acid sequencemay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to convert the RNA to a complementary cDNA. In someembodiments, the RNA is whole cell RNA and is used directly as thetemplate for amplification. Any method known in the art for amplifyingnucleic acid molecules is contemplated (e.g., PCR, LCR, Qbeta Replicase,etc).

Expressed Proteins or Peptides

Genes can be expressed in any number of different recombinant DNAexpression systems to generate large amounts of the polypeptide product,which can then be purified and used in methods and compositions reportedherein. Any method known in the art for generating and using constructsis contemplated. In certain embodiments, genes or gene fragmentsencoding one or more polypeptide may be inserted into an expressionvector by standard cloning or subcloning techniques known in the art.

Proteins, peptides and/or antibodies or fragments thereof may bedetected or analyzed by any means known in the art. In certainembodiments, methods for separating and analyzing molecules may be usedsuch as gel electrophoresis or column chromatography methods.

Electrophoresis

Electrophoresis may be used to separate molecules (e.g., large moleculessuch as proteins or nucleic acids) based on their size and electricalcharge. There are many variations of electrophoresis known in the art. Asolution through which the molecules move may be free, usually incapillary tubes or it may be embedded in a matrix or other materialknown in the art. Common matrices can include, but are not limited to,polyacrylamide gels, agarose gels, mass spec, blotting and filter paper.

Some embodiments, using a gene or gene fragment encoding a polypeptidemay be inserted into an expression vector by standard subcloningtechniques. An expression vector may be used which produces therecombinant polypeptide as a fusion protein, allowing rapid affinitypurification of a peptide or protein. Examples of such fusion proteinexpression systems are the glutathione S-transferase system (Pharmacia,Piscataway, N.J.), the maltose binding protein system (NEB, Beverly,Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6×His system(Qiagen, Chatsworth, Calif.).

Pharmaceutical Formulations

Any pharmaceutical formulation known in the art for a vaccine iscontemplated herein. In certain embodiments, a formulation can containone or more dengue virus serotype in various ratios in a single vaccine.It is contemplated that formulations can contain other agents of use invaccination of a subject including, but not limited to other active orinactive ingredients or compositions known to one skilled in the art.

All contemplated vaccinal viruses herein can be administered in the formof vaccinal compositions which can be prepared by any method known toone skilled in the art. In certain embodiments, the virus compositionsare lyophilized and are mixed with a pharmaceutically acceptableexcipient (e.g. water, phosphate buffered saline (PBS), wetting agentsetc.) In other embodiments, vaccine compositions can include stabilizersthat are known to reduce degradation of the formulation and prolongshelf-life of the compositions.

In other embodiments, an adjuvant may be added to the composition toinduce, increase, stimulate or strengthen a cellular or humoral immuneresponse to administration of a vaccination described herein. Anyadjuvant known in the art that is compatible with compositions disclosedherein is contemplated.

Some embodiments herein concern amounts or doses or volumes ofadministration of a tetravalent dengue virus composition and the amountor dose can depend on route of administration and other specificationssuch as the subject getting the vaccine (e.g. age, health condition,weight etc.).

It is contemplated herein that compositions described can beadministered to a subject living in an area having dengue virus, asubject traveling to an area having dengue virus or other subject suchas any human or animal capable of getting dengue fever or other denguevirus condition. In certain embodiments, it may be recommended that asubject traveling to an area having dengue virus is administered one ormore vaccine compositions (e.g. two or more on Day 0) about 1 to about 3months prior to dengue virus exposure. Vaccines herein can beadministered as a prophylactic treatment to prevent infection in adultsand children. A subject can be naïve or non-naïve subject with respectto exposure to dengue virus and vaccine regimens disclosed herein.

Kits

Other embodiments concern kits of use with the methods (e.g. methods ofapplication or administration of a vaccine) and compositions describedherein. Some embodiments concern kits having vaccine compositions of useto prevent or treat subjects having been exposed or suspected of beingexposed to one or more dengue viruses. In certain embodiments, a kit maycontain one or more than one formulation of dengue virus serotype(s)(e.g. attenuated vaccines, trivalent or tetravalent formulations,DENVax™) at predetermined ratios. Kits can be portable, for example,able to be transported and used in remote areas such as militaryinstallations or remote villages in dengue endemic areas. Other kits maybe of use in a health facility to treat a subject having been exposed toone or more dengue viruses or suspected of being at risk of exposure todengue virus.

Kits can also include a suitable container, for example, a vessel,vials, tubes, mini- or microfuge tubes, test tube, flask, bottle,syringe or other container. Where an additional component or agent isprovided, the kit can contain one or more additional containers intowhich this agent or component may be placed. Kits herein will alsotypically include a means for containing the agent (e.g. a vessel),composition and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow-moldedplastic containers into which the desired vials are retained.Optionally, one or more additional agents such as immunogenic agents orother anti-viral agents, anti-fungal or anti-bacterial agents may beneeded for compositions described, for example, for compositions of useas a vaccine against one or more additional microorganisms.

In other embodiments, kits can include devices for administering one ormore vaccination to a subject such as an ID, SQ, IM, an inhaler,intranasal applicator or other device for administering a vaccinecomposition disclosed herein.

In other embodiments, a single vaccine composition of at least threeserotypes of live, attenuated dengue virus, or fragments thereof for usein rapidly inducing an immune response in a subject against at leastthree dengue virus serotypes, wherein at least two doses of the singlevaccine composition are to be administered in two or more anatomicallocations on the same day of a subject in need thereof, inducingneutralizing antibodies in the subject against at least three denguevirus serotypes. In certain embodiments, the single vaccine compositioncan contain at least one additional booster administration of aformulation of a live, attenuated dengue vaccine is to be administered 1to 180 days after the simultaneous administrations. In otherembodiments, the single vaccine composition can include a predeterminedratio of monovalent vaccines for the three or more dengue virusserotypes in the single vaccine composition. In a single vaccinecomposition, wherein the single vaccine composition comprises equivalentratios of monovalent vaccines for three or more dengue virus serotypesin the single vaccine composition.

A single vaccine composition can include the formulation of the live,attenuated dengue vaccine for the at least one additional boosteradministration can be the same or a different formulation as the firstformulation. If different than the first formulation, a vaccinecomposition can include a pre-determined concentration of one or moremonovalent vaccines for the dengue virus serotypes. Further,concentration of dengue virus serotypes can include a higherconcentration of one or more dengue virus serotype than the formulationused for the same day administrations, wherein the higher concentrationis 2 to 100,000 fold greater concentrations than that used in the singleformulation first administered. In accordance with these embodiments,two or more anatomical locations comprise different anatomical locationsusing the same mode of administration. Two or more anatomical sites caninclude different anatomical locations using different modes ofadministration. A composition can include a tetravalent single vaccinecomposition that represents all four dengue virus serotypes. Inaccordance with these embodiments, a single vaccine composition caninclude all four dengue virus serotype(s) at a predetermined ratio. Thelive, attenuated dengue viruses can include one or more dengue-denguechimeric viruses further comprising capsid and non-structural proteinsof the attenuated dengue virus and pre-membrane and envelope proteins ofat least a second dengue virus. The capsid and non-structural proteinsare from an attenuated dengue-1, dengue-2, dengue-3 or dengue-4 virus.

Some embodiments include a kit of one or more of the above referencedcompositions and one or more device for administration by any modecontemplated herein.

The following examples are included to demonstrate certain embodimentspresented herein. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered to function well in the practices disclosedherein. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thecertain embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope herein.

EXAMPLES Example 1

Previous studies revealed that natural infection with each DENY (denguevirus) serotype leads to long-lived protection against dengue fevercaused by the homologous serotype. In certain embodiments,administration of an effective dengue vaccine closely mimics naturalinfection and can serve as a mode for administering vaccines againstDengue virus. Embodiments reported herein can concern a naturalinfection route of dengue virus (DENY) infection, similar to intradermaldelivery by the transporting host, a mosquito bite. In certainembodiments, intradermal injection to deposit the vaccine viruses intothe same tissue can be used. Skin is a highly accessible organ andrepresents an effective immune barrier, mainly attributed to thepresence of Langerhans cells (LCs) residing in the epidermis. Skinimmunization elicits a broad range of immune responses, includinghumoral, cellular, and mucosal and has the potential to bypass theeffect of pre-existing immunity on the immunogenicity of administeredvaccines.

Some embodiments for intradermal (ID) administration of the tetravalentdengue vaccines in a subject in need of such a treatment are reported.One exemplary method of intradermal administration was performed on fourCynomologous macaques administered a DENVax™ ((DENVax-1: 1×10⁵PFU,DENVax-2; 1×10⁵ PFU, DENVax3: 1×10⁵PFU, DENVax4: 1×10⁵PFU) Dengue virusvaccine) by intradermal administration. To achieve an equivalent dose ofvirus, 0.15 ml of vaccine was deposited ID in three closely spaced sitesusing a needle-free jet injector (see FIGS. 1 and 2, below). FIG. 1represents an intradermal inject (e.g., PharmaJet® or other intradermaldevice) device used for intradermal inoculations.

FIG. 2 illustrates inoculation sites on Cynomolgus macaques postvaccination with PharmaJet device. Animals were boosted 60 days laterwith the same formulation by the same route. Serum samples werecollected at predetermined intervals, days 15, 30, 58, 74, and 91 andwere tested for the presence of neutralizing antibodies directed againstthe four Dengue serotypes. PRNT (plaque reduction neutralization test,known in the art for quantifying levels of anti-DEN neutralizingantibodies) were performed on the sera samples.

It was demonstrated that the neutralizing antibody titers aresignificantly higher after ID administration as compared to SCadministration (p<0.05 for DENY-1 and DENY-2) after a primaryadministration (see FIG. 3) or after a secondary administration (p<0.05for all DENY serotypes) (See FIG. 4). Since the sites were closelyspaced in the same area, and each innoculum consists of all fourviruses, this mode of vaccine delivery closely resembles a singleadministration of DENVax™. FIG. 3 illustrates 50% PRNT (plaque reductionneutralization titers) Geometric Mean Titers at Day 58 (58 days afterthe primary administration). FIG. 4 illustrates 50% PRNT Geometric MeanTiters at Day 74 (14 days after the secondary administration on Day 60).As can be seen in the figures, the neutralizing antibody titers to allfour dengue viruses were higher after intradermal versus subcutaneousadministration. In addition, the number of animals that demonstratedneutralizing antibody responses (“seroconversion” defined as PRNT>10)was greater after the first dose of vaccine (see Table 1, the percentageof animals that seroconverted to each of the four Dengue serotypes isshown after primary and secondary immunization).

TABLE 1 Seroconversion of non-human primates after dengue immunization %Seroconversion DENVax DEN-1 DEN-2 DEN-3 DEN-4 Formulation Prime BoostPrime Boost Prime Boost Prime Boost 5:5:5:5 SC 87.5% 100.0% 100.0%100.0% 75.0% 100.0% 50.0% 100.0% 5:5:5:5 ID 100.0% 100.0% 100.0% 100.0%100.0% 100.0% 100.0% 100.0%

The immunized animals were tested for protection against challenge withwild type dengue viruses. In cynomolgus macaques, wild type dengue virusinfection leads to virus replication and viremia, but no clinical signs.At day 91, two monkeys were challenged with DENY-1 (Dengue virusserotype 1) and two monkeys challenged with DEN-2 (Dengue virus serotype2). Serum samples were collected daily for 11 days after challenge.Levels of dengue virus RNA were measured in the samples by quantitativereal-time polymerase chain reaction technology (q-rtPCR) and titers ofviable virus were measured by virus isolation and plaque formation onVero cells. The results are shown in Tables 2 and 3. Neutralizingantibodies against DEN-1 at Day 91, just prior to challenge(“Pre-Challenge”) and Day 105, 14 days after challenge (“Post”). Viremiais given as the number of days that live DEN-1 virus could be isolatedfrom blood samples (“Duration”) and the log 10 of the peak titerisolated from each animal Viral RNA is given as the number of days viralRNA could be detected in the serum samples (“Duration”) and peak viralRNA levels in each monkey, expressed as the log 10 of the number ofviral RNA genomes detected.

TABLE 2 Responses after challenge with DEN-1 DEN-1 PRNT Pre- ViremiaViral RNA Monkey Formulation Challenge Post Duration Peak Duration PeakCY0174 5:5.5:5 SC 240 240 0 0 0 0 CY0181 5:5:5:5 SC 640 61440 0 0 5 5.6CY0192 5:5:5:5 ID 1920 1280 0 0 0 0 CY0194 5:5:5:5 ID 7680 1920 0 0 0 0CY0061 Controls 1 2560 6 2.0 9 5.7 CY0193 Controls 25 2560 3 2.7 7 6.4CY0058 Controls 1 640 5 2.9 7 5.5 CY0073 Controls 1 1280 5 3.6 10 6.2

TABLE 3 Responses after challenge with DEN-2 DEN-2 PRNT Pre- ViremiaViral RNA Monkey Formulation Challenge Post Duration Peak Duration PeakCY0172 5:5:5:5 SC 3413 3413 0 0 1 3.9 CY0177 5:5:5.5 SC 853 533 0 0 0 0CY0198 5-5:5:5 ID 240 320 0 0 0 0 CY0201 5:5:5:5 ID 1920 1600 0 0 0 0CY0088 Controls 6 10240 6 2.3 8 5.1 CY0199 Controls 1 3840 5 1.8 9 4.7CY0065 Controls 1 10240 5 2.9 8 5.8 CY0104 Controls 1 10240 4 2.4 8 5.7

After challenge, the SC and ID immunized animals were completelyprotected from DEN-1 or DEN-2 induced viremia (compared to the controlanimals that demonstrated significant viremia of long duration). In allof the ID immunized animals, but not all of the SC immunized animals,there was also an absence of viral RNA replication and a lack of anincrease in antibody titer after challenge (compare the ID animals to SCinjected CY0181, CY0172 or the control animals). These data suggest thatprotection is “sterilizing” and prevents any virus replication afterchallenge.

Example 2

In another example, an optimized DENVax™ formulation delivered indifferent locations and with different timings will be tested innon-human primates. Groups of eight Cynomolgus macaques will beimmunized with a DENVax™ formulation containing 1×10⁵ plaque formingunits (pfu), 1×10⁴ pfu, 1×10⁵ pfu and 1×10⁵ pfu of DENVax™-1, DENVax™-2,DENVax™-3 and DENVax™-4, respectively (abbreviated 5:4:5:5). Two doseswill be administered in 0.1 ml ID. Groups will be immunized with eitherone dose in each arm at Day 0, one dose in one arm at Day 0 and one dosein the other arm at Day 7, or one dose in one arm at Day 0 and one dosein the other arm at Day 60. These groups will be compared to a groupthat receives the same dose (5:4:5:5) in three sites in the same are onDay 0 and three sites in the other arm on Day 60 as well as a group thatreceives the same dose in a single 0.5 ml SC immunization in one arm atDay 0 and in the other arm at Day 60. A control group will be immunizedwith vaccine excipients only (no vaccine viruses). Followingimmunization, blood samples will be collected on days 0, 7 (for peakviremia), 15, 30, 60, and 90 to test the neutralizing antibodies againstthe four Dengue virus serotypes by PRNT50. PBMCs collected on days 30,60, 90 will be also monitored for IFN-γ secretion by an ELISPOT assay.On day 90, two animals from each group will be challenged with wild typeof DEN-1, DEN-2, DEN-3, or DEN-4 viruses. Challenged animals will bemonitored for clinical signs and temperature (twice daily), changes infood consumption (once daily) and body weight (weekly). In addition, allanimals will be bled daily for 11 days post-challenge to monitor viremiaand hematological parameters. Again, the speed and duration of PRNTresponses to all four DEN viruses and protection after day 90 challengewill be assessed. It is believed that intradermal administration inmultiple sites and in distinct anatomical locations may be moreeffective than subcutaneous administration as a single bolus. Multiplesites can provide exposure of the vaccine to more antigen presentingcells. Distinct anatomical locations can permit vaccine access tomultiple lymph nodes. In addition, booster immunizations of Denguevaccines have only been administered after the development of antibodyresponses in mice, primates and human clinical trials, thirty days orlonger. At this time, neutralizing antibodies inhibit the response tothe live viral vaccines. It was previously shown that boosting primatesone month after primary immunization was less effective than dosing fourmonths after primary immunization. It was speculated that high levels ofhomologous and heterologous antibodies that circulate after the initialimmunization can inhibit viral replication in a second dose. Whileprolonged (two months or longer) immunization may circumvent thisinhibition, it has not been tested whether accelerated immunizationregimen with shorter immunization intervals, before the development ofpotent neutralizing antibody responses may be advantageous. Such ashortened regimen may be an advantage in endemic countries or fortravelers, where exposure to Dengue viruses in between the immunizationsmay put them at risk of disease.

Example 3

In another example, a human clinical trial has been initiated, studyingthe safety and immunogenicity of two DENVax™ formulations, administeredin 0.1 ml either by ID or SC injection. Groups of 12 individuals will beimmunized with for example, a low dose DENVax™ formulation (8×10³ pfu,5×10³ pfu, 1×10⁴ pfu and 2×10⁵ pfu of DENVax™-1, -2, -3 and -4,respectively) or a high dose (2×10⁴ pfu, 5×10⁴ pfu, 1×10⁵ pfu and 3×10⁵pfu of DENVax™-1, -2, -3 and -4, respectively) of DENVax™ ID or SC onDays 0 and 90. Two control groups will be injected SC or ID withphosphate-buffered saline. Patients will be monitored for any adverseevents, and for any significant changes in hematological or bloodchemistry parameters. Serum samples will be collected to measure vaccinevirus replication and neutralizing antibody responses at periodicintervals.

Example 4

Immunogenicity and efficacy of DENVax™ administered intradermally inAG129 mice. In another example, two studies were performed to comparethe effect of route of administration on immunogenicity and efficacy ofDENVax™ in AG129 mice. In one example, the immunogenicity of monovalentDENVax™-4 (e.g. vaccine against one Dengue virus serotype) was comparedin AG129 mice by measuring the neutralizing antibody responses followingSC injection under the skin on the back or ID injection into the footpad using a needle and syringe. Groups of 8 AG129 mice were injected IDor SC with 10⁵ PFU/dose of chimeric DENVax™-4 vaccine in 50 μl and 100μl final volume, respectively. Six weeks after priming, animals fromeach treatment group were boosted via the corresponding ID or SC routewith 10⁵ PFU of DENVax™-4 or TFA. Mice were bled on Day 31 and 58 andcollected sera were pooled to measure neutralizing antibody responses.

Immunization of DENVax™-4 via the ID route elicited a 5-fold higherneutralizing antibody response to DEN-4 after the boost compared to theresponse induced via the SC route (see for example, FIG. 4). Theanti-DEN-4 response elicited by either route of immunization had amarked cross-neutralizing activity against DEN-3 but not against DEN-1or DEN-2 serotypes. FIG. 4 represents neutralizing antibody responsesfollowing primary and secondary immunization of AG129 mice with chimericDENVax™-4. Mice were bled on Day 31 and 58 and collected sera werepooled to measure neutralizing antibody responses using the plaquereduction assay (PRNT50).

Two weeks after the boost animals from each group were split in to twogroups and challenged with 10⁶PFU of DEN-1 (Mochizuki virus strain) orDEN-2 (New Guinea C strain) viruses. Challenged animals were monitoredfor clinical signs of disease and survival rates were recorded over aperiod of 5 weeks. Mice immunized via the ID route showed no signs ofdisease after DEN-1 challenge (FIG. 5A). In the SC immunized group onlyone mouse succumbed to infection while the rest of animals had no anyapparent signs of infection (FIG. 5B). In contrast, all control animalssuccumbed to infection by day 13 after DEN-1 challenge (FIG. 5A).Following DEN-2 challenge, all animals immunized with only DENVax™-4 viathe SC route succumbed to infection by day 25 with mean survival time(MST) of 19.5 days as compared to the control (FTA) mice that allsuccumbed by day 17 (MST=12.5 days) post-challenge (FIG. 5B). Incontrast, fifty percent of ID DENVax™-4 immunized mice survived theinfection until the end of the 5 week monitoring period (FIG. 5B). FIGS.5A and 5B represent survivals of DENVax™-4 immune AG129 mice followingchallenge with DEN-1 (a) or DEN-2 (b) viruses. Challenged animals weremonitored for clinical signs of disease and survival rates were recordedover a period of 5 weeks.

In a second study, immunogenicity of tetravalent DENVax™ vaccineadministered SC or ID in mice (e.g. AG129) was tested. Groups of AG129mice, six per group were injected SC or ID with the DENVax™ in 100 μl or50 μl (final volume), respectively. Mice were immunized with DENVax™ ata 5:4:5:5 (10⁵ PFU of DENVax™-1, -3 and -4 and 10⁴ PFU of DENVax™-2)dose level of composite chimeric vaccines. All immunized animalsreceived a booster injection of 5:4:5:5 DENVax™ (10⁵ PFU of DENVax™-1,-3 and -4 and 10⁴ PFU of DENVax™-2) 42 days' post-primary inoculation.Blood samples were collected on days 42 and 56 to measure neutralizingantibody responses to each DEN virus serotype.

As represented in Table 4, both primary and secondary neutralizingantibody responses to all four DEN serotypes were induced. Following theboost, the neutralizing anti-DEN-1, DEN-3 and DEN-4 antibody titers wereincreased by 2, 5 and 2 fold, respectively in the group of mice injectedID as compared to the SC immunized animals. Neutralizing responses toDEN-2 virus were comparable in both groups Immunization via the SC routeresulted in a profile of dominant neutralizing antibody responsesagainst DEN-1>DEN-2>DEN-3>DEN-4, with neutralizing titers 5120, 1280,640 and 80, respectively. The hierarchy of neutralizing antibodyresponses after ID administration had shifted as follows;DEN-1>DEN-3>DEN-2>DEN-4 with neutralizing antibody titers 10240, 3840,1280 and 160, respectively.

TABLE 4 Comparison of the immunogenicity of tetravalent DENVax ™ bearingthe ratio 5:4:5:5 PFU of each composite chimeric virus (10⁵ PFU ofDENVax ™-1, -3 and -4 and 10⁴ PFU of DENVax ™-2) after SC or IDimmunization of mice. Blood samples were collected on days 42 and 56 tomeasure neutralizing antibody responses to each DEN virus serotype.Neutralizing Antibody Titers (GMT) DENVax ™ DEN-1 DEN-2 DEN-3 DEN-4Formulation Prime Boost Prime Boost Prime Boost Prime Boost 5:4:5:5/SC1920 5120 3200 1280 1280 640 80 80 5:4:5:5/ID 2560 10240 1280 1280 16003840 120 160

Materials and Methods

Mice: AG129 mice have an “intact” immune system; deficient for theinterferon (IFN)-α/β and -γ receptors. Dengue infection has beendescribed for this model. Other studies: pathogenesis, cell tropism, andADE have also been examined. This model permits challenge with DEN-1 andDEN-2.

Nonhuman primates: Cynomolgus, rhesus macaques carry virus (viremia),but no disease manifests.

Rapid Dosing Study Example 5

In one exemplary study, immune responses to tetravalent Dengue vaccineswere evaluated for different routes of administration and dosingregimens in the non-human primate model comparing vaccine delivery byconventional needle injection to needle-free administration. Thequantifiable endpoints for the nonhuman primate study are i) the routefor greatest geometric mean neutralizing antibody titer against each ofthe four dengue serotypes in non-human primates and ii) the protectionfrom challenge with two of the dengue serotypes.

Two dosing schedules were evaluated in this study—two consecutive doseson Day 0 (at different anatomical sites) were compared to administrationof two doses given 60 days apart (0.60). The high dose formulation ofthe tetravalent formulation (e.g. DENVax™) was used for immunization inthis study. This vaccine lot is the same material used for two Phase 1studies being conducted. The high dose tetravalent formulation vaccineconsists of 2×10⁴ pfu of DEN-1, 5×10⁴ pfu of DEN-2, 1×10⁵ pfu DEN-3 and3×10⁵ pfu DEN-4. The study design for the nonhuman primate study isshown in Table 5.

TABLE 5 Nonhuman Primate Study Route/ No. of Challenge Method ofImmuniza- Site(s) for on Day 90, Adminis- Group Treatment tions DosingSC route tration 1 High dose Day 0 Two 3 animals ID DENVax (both arms)with wt PharmaJet DENV-2, Injector 3 animals with wt DENV-4 2 High doseDay 0, One 3 animals ID DENVax Day 60 (alternate with wt PharmaJet arms)DENV-2, Injector 3 animals with wt DENV-4 3 High dose Day 0, One 3animals ID DENVax Day 60 (alternate with wt Needle/ arms) DENV-2,Syringe 3 animals with wt DENV-4 4 High dose Day 0 Two 3 animals SCDENVax (both arms) with wt PharmaJet DENV-2, Injector 3 animals with wtDENV-4 5 High dose Day 0, One 3 animals SC DENVax Day 60 (alternate withwt PharmaJet arms) DENV-2, Injector 3 animals with wt DENV-4 6 High doseDay 0, One 3 animals SC DENVax Day 60 (alternate with wt Needle/ arms)DENV-2, Syringe 3 animals with wt DENV-4 7 PBS Day 0, One 3 animals IDDay 60 (alternate with wt PharmaJet arms) DENV-2, Injector 3 animalswith wt DENV-4

Serum samples were collected after each vaccination and wild type denguevirus challenge on Days 0, 3, 5, 7, 10, 12, 14, 53, 64, 67, 88, 91, 93,95, 97, 99, 101, 102 and 104 to analyze the samples for dengue viremia.Serum samples were also collected on Days 0, 30, 53, 75, 88 and 104 todetermine the levels of neutralizing antibodies induced by thetetravalent formulation administered by needle/syringe or the IDinjector.

Serum samples were collected at specified intervals during the course ofthe study. Sera collected on Days 0, Day 30 and Day 88 (pre-boost) havebeen assayed for neutralizing antibodies to Dengue-1, Dengue-2, Dengue-3and Dengue-4. The GMT antibody titers are shown below in Table 6.

TABLE 6 Neutralizing antibody titers to all four dengue serotypes forDays 30, 53, 75 and 88 after one or two immunizations with DENVax. DoseSchedule/ Route/Method of Day 30 Post-Dose 1, Day 53 Post-Dose 1,Administration/ Reciprocal GMTs Reciprocal GMTs Group Treatment DEN-1DEN-2 DEN-3 DEN-4 DEN-1 DEN-2 DEN-3 DEN-4 1 2 doses (Day 0), 80 1280 12736 63 1280 40 13 PJ ID, DENVax 2 2 doses (Day 0, 60), 18 14 101 11 32 1422 10 PJ ID, DENVax 3 2 doses (Day 0, 60), 160 36 64 9 45 40 10 5 N/SID, DENVax 4 2 doses (Day 0), 1016 1016 403 80 640 1280 127 45 PJ SC,DENVax 5 2 doses (Day 0, 60), 154 1816 226 64 285 1280 57 22 PJ SC,DENVax 6 2 doses (Day 0, 60), 80 1140 113 11 80 806 20 28 N/S SC, DENVax7 2 doses (Day 0, 60), 10 5 6 5 7 5 5 5 PJ ID, PBS Dose Schedule/Route/Method of Day 75 Post-Dose 1, Day 88 Post-Dose 1, Administration/Reciprocal GMTs Reciprocal GMTs Group Treatment DEN-1 DEN-2 DEN-3 DEN-4DEN-1 DEN-2 DEN-3 DEN-4 1 2 doses (Day 0), 40 806 25 25 57 1016 25 25 PJID, DENVax 2 2 doses (Day 0, 60), 113 28 63 71 160 90 80 45 PJ ID,DENVax 3 2 doses (Day 0, 60), 160 40 57 36 90 101 57 36 N/S ID, DENVax 42 doses (Day 0), 403 718 90 57 254 640 90 64 PJ SC, DENVax 5 2 doses(Day 0, 60), 508 1810 226 113 403 1280 127 80 PJ SC, DENVax 6 2 doses(Day 0, 60), 143 806 71 57 113 4064 32 40 N/S SC, DENVax 7 2 doses (Day0, 60), 5 5 5 5 5 5 5 5 PJ ID, PBS Neutralizing antibody titers of <10are reported as “5”. Serum dilutions started at 1:10 Seroconversion(values in parenthesis) is defined as titer >10 over Day 0 <10 baselinetiter or a >4-fold rise in titer if baseline titer on Day 0 was >10.Serum dilutions for analysis started at 1:10. Results were generatedfrom duplicates or triplicates. PJ = exemplary PharmaJet needle-freeinjector, N/S = needle/syringe; GMT = Geometric mean titer; ID =intradermal; SC = subcutaneous Data are presented as geometric meantiter (GMT) ± standard error (SE)

All 42 animals in the study were seronegative at the start of the studyand displayed no neutralizing antibody titers to any of the four dengueserotypes on Day 0. The results on Day 30 after priming the animals withDENVax™ showed that animals receiving two doses of DENVax™ on Day 0 (onedose in each arm) by either the ID or SC route of administration,displayed a high neutralizing antibody titer to Dengue-1, Dengue-2 andDengue-4 (Groups 1 and 4). Seroconversion rates by day 30 were 100% forboth groups as compared to groups 2 and 3. Both groups maintained highlevels of neutralizing antibody responses up to day 88 just prior tovirus challenge.

For live attenuated vaccines, vaccine virus replication afterimmunization is an important measure of vaccine uptake and vaccinesafety. Vaccine virus replication in the nonhuman primates was evaluatedafter the first and second immunization with a live attenuatedtetravalent formulation vaccine (DENVax™). Serum samples collected onDays 0, 3, 5, 7, 10, 12, 14 after the first immunization were tested forthe presence of viral RNA from the vaccine strains using a qRT-PCR assay(see Table 7).

TABLE 7 DENVax-2 RNA detected in the serum after primary immunizationwith DENVax. No. Animals Positive for Viral RNA, DENVax-2 Viral RNA,Log₁₀ GE/mL Group Dosing Schedule Day 5 Day 7 Day 10 Day 12 Day 14 1 2doses (Day 0), — 3/6 4/6 5/6 3/6 PJ ID (3.9-4.8) (4.5-5.3) (3.9-5.2)(3.8-5.1) 2 2 doses (Day 0, 60), — — — — — PJ ID 3 2 doses (Day 0, 60),— — 1/6 1/6 1/6 N/S ID (3.8) (3.8) (3.8) 4 2 doses (Day 0), 1/6 5/6 5/61/6 — PJ SC (3.8) (3.8-5.0) (3.7-5.3) (4.0) 5 2 doses (Day 0, 60), 1/65/6 5/6 5/6 3/6 PJ SC (3.8) (4.5-5.4) (3.8-5.4) (3.2-4.8) (3.7-5.0) 6 2doses (Day 0, 60), — 3/6 4/6 3/6 2/6 N/S SC (3.9-5.6) (3.8-5.4)(4.3-5.0) (3.7-4.2) 7 2 doses of PBS (Day — — — — — 0, 60), PJ IDResults are averages from duplicate or triplicate data. Samples withtiters <log₁₀ 3.6 were considered negative. GE/mL = genomeequivalents/mL N/S = needle/syringe; PJ = PharmaJet needle-free injector

Viral RNA was not detected on Day 0 (pre-vaccination) and Day 3(post-immunization). For all groups, viral RNA was detected only for theDengue-2 serotype from day 5 to day 14 post-vaccination after the firstimmunization. For Groups 1, 3, 5 and 6 endpoint titers were not observedby 14 days post-immunization. Peak titers were observed on Day 10 forGroups 1 and 4, and on Days 7 and 10 for Groups 5 and 6 (Table 7). ViralRNA was not detected for any of the groups after the second immunizationevaluated on Days 64 and 67 (4 and 7 days post-dose 2).

On Day 90, three animals from each group were challenged with eitherwild-type Dengue-2 or Dengue-4 to demonstrate efficacy upon immunizationwith the tetravalent formulation. Protected animals should exhibit alack of wild-type Dengue virus infection and replication. Wild-typechallenge virus (Dengue-2 and Dengue-4) replication was analyzed for allof the groups after challenge with 106 PFU wild-type Dengue 2 (NewGuinea C strain) and Dengue 4 (814669 strain) viruses on Days 91, 93,95, 97, 99, 101, 102 and 104 (Table 8). Dengue vaccine (e.g. DENVax™.

TABLE 8 Protection of DENVax-immunized NHPs from wt DENV challenge.Pre-Challenge Antibody Post-Challenge Vaccination & Titers (GMT), Day 88Viremia (log₁₀ GE/mL) Challenge Regimen DEN-1 DEN-2 DEN-3 DEN-4 Day 3Day 5 Day 7 2 doses on Day 0, DENV-2 57 1016 25 25 — — — PJ ID, DENVaxDENV-4 — — — 2 doses on Day 0, 60, DENV-2 160 90 80 45 — — — PJ ID,DENVax DENV-4 — — — 2 doses on Day 0, 60, DENV-2 90 101 57 36 — — — N/SID, DENVax DENV-4 — — — 2 doses on Day 0, DENV-2 254 640 90 64 — — — PJSC, DENVax DENV-4 — — — 2 doses on Day 0, 60, DENV-2 403 1280 127 80 — —— PJ SC, DENVax DENV-4 — — — 2 doses on Day 0, 60, DENV-2 113 4064 32 40— — — N/S SC, DENVax DENV-4 — — —

Viral RNA of the wild-type challenge viruses was detected only in Group7 that had received PBS. For Dengue-2, viral RNA was detected in 3 of 3animals on Days 93 to 97. For Dengue-4, viral RNA was detected in only 1of 3 animals on Day 95. One important observation of the groups thatwere immunized with the tetravalent formulation is that no viral RNA foreither the Dengue-2 or the Dengue-4 challenge viruses was observed.These results suggested that the tetravalent formulations immunizationby any of the dosing schedules tested conferred immune protectionagainst challenge of both Dengue-2 and Dengue-4 wild-type viruses.

Overall, this nonhuman primate study clearly showed that the noveldosing schedule of administering two doses of a tetravalent formulationon Day 0 at two distinct sites (e.g. different arms) induced levels ofneutralizing antibodies that were equivalent or higher than thoseobserved for more traditional dosing schedules of delivering the primeand boost immunization 2 to 3 months apart. The onset of the immuneresponses was more rapid for the groups that received two doses on Day 0and long lasting. The application of the needle-free ID or SC injectorenhanced the immune responses such that higher titers were observed.

Example 6 AG129 Mouse Studies on Rapid Immunization

In another exemplary study, novel dosing schedules were designed thatexplore either administration of two vaccine doses at two distinct siteson a single occasion or shorter dosing intervals between two doses ofvaccine which will enhance compliance of vaccinated subjects to returnfor the second immunization. Standard Dengue vaccines developedpreviously typically require three doses over the course of a year toachieve robust multivalent Dengue immune responses. With respect tovaccination schedules presented herein, response was evaluated forimmunization occurring in at least two anatomical two sites, andadministering, in certain embodiments, a full dose (see Table 9) at eachsite intradermally. This protocol was performed in part to activateimmune cells and antigen presenting cells in two different lymph nodeson Day 0 to induce higher levels and more robust dengue-specific immuneresponses compared to administering two doses intradermally 7, 14 or 42days apart. In one study two routes of administration were compared, SCand ID routes using a conventional 42-day interval between vaccinations.The mice were immunized with a low dose formulation of a tetravalentformulation (DENVax™; 3:3:3:3 ratio of each of the serotypes) whichconsisted of 10³ PFU of each Dengue-1, -2, -3, and -4 (e.g. DENVax™-1,-2, -3 and -4) in a 0.05 mL volume given via the intradermal route (inthe foot pad). The in live portion of this study was conducted prior toinitiation of this contract. The study design is shown in Table 9 below.

TABLE 9 Study design for AG129 mouse study DEN-012. Number of Number ofGroups Dose Immunizations/Route Animals A DENVax ™ 1 (day 0)/ID 6(3:3:3:3) B DENVax ™ 2 (day 0)/ID, giving a 6 (3:3:3:3) full dose intoeach of two footpads C DENVax ™ 2 (7 days apart)/ID 6 (3:3:3:3) DDENVax ™ 2 (14 days apart)/ID 6 (3:3:3:3) E DENVax ™ 2 (42 daysapart)/ID 6 (3:3:3:3) F FTA 2 (14 days apart)/ID 6 (negative controlgroup) G DENVax ™ 2 (42 days apart)/SC 6 (3:3:3:3)

The neutralizing antibody titers to Dengue 1-4 present in the collectedmouse sera were determined by a microneutralization assay. Sera werecollected at specified time points throughout the study and thelongevity of the immune responses was studied by maintaining the studygroups until Day 160 (longer than 5 months after study start). Theresults obtained from sera collected on Days 28 and 56 post-immunizationare illustrated in Table 10.

TABLE 10 Neutralizing antibody titers (GMTs) to DEN-1, -2, -3 and -4GMT, Day 28 Group Treatment Groups DEN-1 DEN-2 DEN-3 DEN-4 A 1 (day0)/ID 400 100 200 40 B 2 (day 0)/ID, giving 800 200 800 160 a full doseinto each of two footpads C 2 (42 days apart)/ID 400 100 200 40 D 2 (14days apart)/ID <20 <20 <20 <20 (negative control) GMT, Day 56 GroupDEN-1 DEN-2 DEN-3 DEN-4 A 800 200 400 40 B 3200 400 1600 160 C 1600 200800 40 D <20 <20 <20 <20

In previous studies, a conventional dosing schedule of priming animalswas used on Day 0 and then administering a booster vaccination on Day 42to evaluate the immune responses for the tetravalent dengue vaccine.Both prime and boost vaccinations were administered by the subcutaneous(SC) route. This dosing schedule was included in the study forcomparison to the novel dosing schedules. Initially, one study(represented in Table 10) compared the SC and ID routes ofadministration using the conventional dosing interval of giving twodoses 42 days apart. The results indicate that there is no significantdifference between the SC and ID routes with respect to neutralizingantibodies induced in this mouse model. This study further exploredwhether two doses administered on Day 0 at two anatomical sites (onedose into each of two foot pads) could induce neutralizing antibodylevels similar to the standard dosing schedule (2 doses 42 days apart)described above. The results show that immunization on Day 0 at twosites each, with a full dose of a tetravalent formulation of DENVax™,via the ID route induced neutralizing antibody levels to all four dengueserotypes that are equivalent in magnitude to the conventional dosingschedule. The effect of a single vaccine dose administered by the IDroute was also studied (Group A). Administration of a single dose ofDENVax™ on Day 0 resulted in antibody responses that trended slightlylower compared to two doses on Day 0 (compare Groups A and B).Increasing the interval between the two doses from 7 to 42 days didincrease antibody responses beyond the levels observed. Evaluation ofthe longevity of the Dengue immune response revealed that neutralizingantibody titers to all four dengue serotypes remained at high levels atDay 160 post-immunization independent of route of administration anddosing schedule (data not shown).

Overall, the results suggested that the intradermal route ofadministration induces neutralizing antibody levels equivalent to thoseobserved for the subcutaneous route. Further, the administration of twodoses on Day 0 at two different sites by the ID route induced a robustneutralizing antibody response equivalent to conventional dosingschedules. The antibody responses induced were long lasting anddecreased only slightly. The animals did not display increased morbidityand mortality. This study demonstrated that administration of twovaccine doses at two distinct sites is a viable option for immunizationas the resulting antibody titers and duration of immune responses areequivalent in magnitude to those resulting from two doses given 42 daysapart. These dosing regimens will be beneficial for travelers to dengueendemic regions and others in need of fast protection from dengue virusexposure.

Example 7 Another Rapid Immunization Study in AG129 Mice

The objective of this study was to determine whether administering twodoses at two sites ID on Day 0 will induce higher levels and more robustDengue-specific immune responses compared to administering two doses ID42 days apart. The hypothesis to be tested was whether administration ofa full vaccine dose to each of two sites intradermally will activateimmune cells and antigen presenting cells that traffic to two differentlymph nodes, thereby reducing interference between the four DENVax™vaccine components. The design for this AG129 mouse study is shown belowin Table 11.

TABLE 11 Design of Example 7 AG129 mouse study Number of Number of GroupDose Immunizations/Route Animals 1 DENVax ™ 2 doses (day 0)/ID usingboth 8 3:3:3:3 footpads 2 DENVax ™ 2 doses (day 0, day 42)/ID 8 3:3:3:3using both footpads 3 DENVax ™ 2 doses (day 0)/ID using both 8 4:3:4:5footpads 4 DENVax ™ 2 doses (day 0, day 42)/ID 8 4:3:4:5 using bothfootpads 5 FTA 2 doses (day 0)/ID using both 8 (negative footpadscontrol group)

In this exemplary method, two different vaccine dose levels (low andmedium dose), were used for immunization using the novel dosing scheduleof administering two doses on Day 0 compared to two doses 42 days apart.The mice were dosed with either a low dose formulation of DENVax™(3:3:3:3) which consisted of 10³ PFU of each of DENVax™-1, -2, -3, and 4in a 0.05 mL volume given via the intradermal route (in the foot pad) ora medium dose formulation of DENVax™ (4:3:4:5) which contained 10⁴ PFUof DENVax™-1, 10³ PFU of DENVax™-2, 10⁴ PFU of DENVax™-3, and 10⁵ PFU ofDENVax™-4 in a 0.05 mL volume. On Day 0 all mice were immunized andGroups 2 and 4 were boosted on Day 42. Sera for antibody analysis werecollected on Days 14, 41 and 56 post-primary vaccination and analyzedusing a plaque reduction microneutralization assay to determine theneutralizing antibody levels to all four dengue serotypes Immunogenicityresults obtained from pooled mouse serum samples are shown in Table 12.

TABLE 12 Neutralizing antibody titers for AG129 mouse study DEN-013Reciprocal Neutralizing Antibody Titers (PRNT50) 14 Days p.i.¹ 41 Daysp.i.¹ 56 Days p.i.¹ Group DEN-1 DEN-2 DEN-3 DEN-4 DEN-1 DEN-2 DEN-3DEN-4 DEN-1 DEN-2 DEN-3 DEN-4 1 320 160 80 20 640 640 640 80 800 1600800 40 2 320 320 40 20 640 640 320 80 800 800 400 40 3 640 80 80 40 2560640 1280 160 3200 800 800 40 4 320 80 40 20 320 160 640 80 1600 800 80080 5 20 20 20 20 20 20 20 20 20 20 20 20 ¹p.i.—post-infection

In this example, immunization with either the low or medium dosetetravalent vaccine (e.g. DENVax™) formulation induced neutralizingantibodies to all four dengue serotypes at the early check on Day 14post-vaccination, independent of administration of one vs. two doses onDay 0. The medium dose DENVax™ formulation induced slightly higherneutralizing antibody titers by Day 28 for Groups 1 and 3 particularlyfor DEN-1 and DEN-3, that received two doses on Day 0 compared to groupsthat received only a single dose on Day 0 (Groups 2 and 4). The antibodytiters obtained from sera collected on Day 56 indicate that theneutralizing antibody responses persisted and did not wane regardless ofwhether the animals were boosted on Day 42 or received vaccine only onDay 0. The results obtained in this study further support theapplication of the novel dosing schedule of administering two doses onDay 0 at two distinct sites (e.g. immunologically).

TABLE 13 Neutralizing antibody titers for AG129 mouse study DEN-013.DEN-1 DEN-2 DEN-3 DEN-4 Day 28 Day 56 Day 28 Day 56 Day 28 Day 56 Day 28Day 56 Study 1 3:3:3:3 2 (d 0) 640 800 320 1600 160 800 20 40 2 (d 0,42) 320 800 320 800 160 400 40 40 4:3:4:5 2 (d 0) 1280 3200 320 800 640800 80 40 2 (d 0, 42) 320 1600 160 800 80 800 40 80 FTA 2 (d 0) 40 20<20 20 20 20 <20 20 NMS 20 20 20 20 20 20 <20 20 Study 2 3:3:3:3 2 (d 0)800 3200 200 400 800 1600 160 160 2 (d 0, 42) 400 1600 100 200 200 80040 40 FTA 2 (d 0, 14) 20 <20 <20 <20 20 20 20 <20 NMS 20 20 40 20 20 2020 20

ELISPOT dengue virus neutralizing titers calculated using 50% NMS cutoffat a starting dilution of 1:20. Serum from individual animals within agroup were pooled and tested in triplicate.

Example 8

FIGS. 9A-9D represent graphs comparing neutralizing antibody titersachieved in non-human primates after immunization with tetravalentDENVax containing DENVax-1 (1×10⁵ pfu); DENVax-2 (1×10⁴ pfu); DENVax-3(1×10⁵ pfu); DENVax-4 (1×10⁶ pfu). Two groups were vaccinated with theneedle-free PharmaJet device via the subcutaneous route either twice onthe same day (0,0) or once on day 0 and again on day 60 (0,60). Serumwas analyzed for presence of antibodies on days 0, 30, 53, 75 and 88,and the detection of antibodies against four dengue serotypes wereanalyzed (DEN-1, DEN-2, DEN-3, DEN-4).

In another example, seronegative human subjects were immunized with twodoses of a tetravalent formulation of DENVax containing DENVax-1 (1×10⁴pfu); DENVax-22 (1×10³ pfu); DENVax3 (1×10⁴ pfu); DENVax-4 (1×10⁵ pfu).The route of immunization was subcutaneous or intradermal, and thevaccinations were given 90 days apart. Antibody levels against each ofthe dengue serotypes were analyzed on days 0, 30, 60, 90 and 120. Thevaccine induced neutralizing antibodies to all four serotypes. However,the levels of seroconversion were different when comparing the routes ofimmunization. Overall, the intradermal route of immunization producedappeared to be more “balanced” immune responses in this study, with thelevels of antibodies being more equivalent as compared to thesubcutaneous route.

FIG. 10 represents the data obtained from a human clinical trial inColombia. Seronegative humans were given two doses of a tetravalentformulation of DENVax containing DENVax-1 (1×10⁴ pfu); DENVax-2 (1×10³pfu); DENVax-3 (1×10⁴ pfu); DENVax-4 (1×10⁵ pfu) subcutaneously orintradermally. Antibody levels against each of the dengue serotypes wereanalyzed on days 0, 30, 60, 90 and 120.

In this exemplary method, non-human primates were immunized with twodoses of a tetravalent vaccine (e.g. DENVax™ DENVax-1: 2×10⁴ pfu,DENVax-2: 5×10⁴ pfu, DENVax-3: 1×10⁵ pfu, DENVax-4: 3×10⁶ pfu) eithersimultaneously on Day 0, or two separate doses on days 0 and 60. Thevaccine induced neutralizing antibodies to all four Dengue serotypes. Byday 90 post vaccination, the neutralizing antibody titers of the twogroups were relatively equal (FIG. 11). However, the kinetics of theimmune response was more rapid in the group which received twoimmunizations on day 0. The results obtained in this study furthersupport the application of the novel dosing schedule of administeringtwo doses on Day 0 at two immunologically distinct sites.

FIG. 11 represents a graph comparing neutralizing antibody titersachieved in non-human primates after subcutaneous immunization withtetravalent DENVax containing DENVax-1 (1×10⁵ pfu); DENVax-2 (1×10⁴pfu); DENVax-3 (1×10⁵ pfu); DENVax-4 (1×10⁶ pfu). Two groups werevaccinated either twice on the same day (0,0) or once on day 0 and againon day 60 (0,60). Serum was analyzed for presence of antibodies on days0, 28, 58, 73 and 90, and the detection of antibodies against fourdengue serotypes were analyzed (DEN-1, DEN-2, DEN-3, DEN-4).

Example 9

FIG. 12 represents analysis of single dose administration versus dualadministration in separate anatomical locations of dengue virusvaccines. This data represents changes in levels of various genetranscripts in a subject after 0,0 or single injection where themagnitude of change in gene expression is greater for dualadministration (double dose). The genes included in the clusteranalysis, represented in Cluster 2 include several genes that change inrelation to induction of innate immunity. Thus, induction of innateimmunity and related genes can be greater in a subject having dualadministration at separate anatomical locations of compositionsdisclosed herein. It is likely that the composition induces a responsein multiple anatomical regions (e.g. lymph nodes etc.) reducinginterference and affecting multiple genes that participate in innateimmune responses.

The four dengue virus serotypes (DENV-1-4) are responsible for the mostprevalent mosquito-borne viral illnesses in humans worldwide.Tetravalent vaccines are under development, but up to the instantapplication require multiple immunizations over a period of 6 months toone year. A rapid immunization strategy (RIS) that elicits an immuneresponse to all four DENY serotypes and requires fewer visits to ahealth provider, thus increasing vaccine compliance and inducing rapidseroconversion, would increase safety for people in endemic countries aswell as protect travelers and military personnel from dengue. RISconsisting of two full vaccine doses being administered on the initialvaccination visit (day 0) at two different anatomical locations wasinvestigated using various tetravalent formulations having predeterminedratios. This vaccination strategy resulted in efficient priming andinduction of potent neutralizing antibody responses to all four denguevirus serotypes of long duration (3 months) as compared to thetraditional prime and subsequent 2^(nd) dose (boost) several weeks ormonths later. In addition, analysis of innate immune responses followingprimary immunization support the view that the priming efficiencyafforded by RIS is a result of a higher magnitude of immune signaturesstimulated by this immunization protocol as compared to the single doseimmunization.

Compositions disclosed herein include chimeric dengue virus compositionswhere a backbone of one dengue virus can accommodate one or more of theother dengue virus components. Mixtures of these chimeric compositionscan be used to generate trivalent or tetravalent formulations of use inmethods disclosed herein.

FIGS. 13A-13F represent expression levels over time of various genesassociated with innate immunity in response to single or dualadministration of dengue virus vaccines. This data illustrates thatexpression of certain genes are increased in response to a dualadministration (in separate anatomical locations such as each arm)versus single administration of a vaccine composition disclosed herein(e.g. tetravalent dengue virus composition). Various genes illustratedinclude A. interferon-induced 17 kDa protein (ISG15), a 15-kDa proteinof unique primary amino acid sequence; B. IF144 (interferon-inducedprotein 44); C. XAF1, XAF1 antagonizes the anticaspase activity of XAP1;D. OASL (The human 2′,5′-oligoadenylate synthetase-like gene (OASL)encoding the interferon-induced 56-kDa protein); E. MX1 isinterferon-induced GTP-binding protein Mx1 is a protein that in humansis encoded by the MX1 gene and F. IFIT1, IFN induced protein withtetratricopeptide repeat 1. Therefore, this method of administrationcould be used to rapidly induce innate immune response in a subject inneed thereof such as a tourist visiting a dengue endemic country.

TABLE 14 Gene Fold Rank symbol Gene name change 1 IFI44 IFN-inducedprotein 44 2.96 2 DDX58 DEAD (Asp-Glu-Ala-Asp) box 1.95 polypeptide 58 3IFIT1 IFN-induced protein with 1.90 tetratricopeptide repeat 1 4 OASL2′-5′-oligoadenylate 1.79 synthetase-like 5 MX1 Myxovirus (influenzavirus) 1.77 resistance 1, IFN-inducible 6 ISG15 ISG15 ubiquitin-likemodifier 1.76 7 XAF1 XIAP-associated factor 1 1.72 8 SECTM1 Secreted andtransmembrane 1 1.65 9 IFI27 Interferon, alpha-inducible 1.62 protein 2710 EIF2AK2 Eukaryotic translation initiation 1.59 factor 2-alpha kinase2 11 OAS2 2′,5′-oligoadenylate synthetase 1.59 12 IFI6 Interferon,alpha-inducible protein 6 1.47 13 RNF213 Ring finger protein 213 1.45

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the methods described hereinwithout departing from the concept, spirit and scope herein. Morespecifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept as defined bythe appended claims.

What is claimed: 1-24. (canceled)
 25. A method for inducing an immuneresponse in a subject against three or more dengue virus serotypes, themethod comprising: administering a first immunogenic composition ofthree or more live, attenuated dengue viruses, the compositioncomprising at least one non-chimeric live, attenuated dengue-2 virus andat least one dengue-dengue chimera having a live, attenuated dengue-2virus backbone; and administering a second immunogenic composition ofthree or more live, attenuated dengue viruses, the compositioncomprising at least one non-chimeric live, attenuated dengue-2 virus andat least one dengue-dengue chimera having a live, attenuated dengue-2virus backbone; wherein the first immunogenic composition and the secondimmunogenic composition are administered at the same anatomical locationon the same day to the subject; and inducing an immune response in thesubject against the three or more dengue virus serotypes.
 26. The methodof claim 25, wherein the first immunogenic composition and the secondimmunogenic composition comprise different compositions.
 27. (canceled)28. The method of claim 25, wherein the first immunogenic compositioncomprises three or four dengue virus serotypes at predetermined ratios.29. The method of claim 25, wherein the first immunogenic compositioncomprises a tetravalent formulation of all four dengue virus serotypes.30-31. (canceled)
 32. The method of claim 25, wherein the at least onedengue-dengue chimera of a live, attenuated dengue virus comprisesnon-structural proteins from at least one dengue virus serotype andpre-membrane and envelope proteins from at least a second dengue virusserotype.
 33. The method of claim 25, wherein the at least onedengue-dengue chimera of a live, attenuated dengue virus is selectedfrom dengue-2/dengue-1, dengue-2/dengue-3, and dengue-2/dengue-4,wherein dengue-2 is the backbone and dengue-1, dengue-3 or dengue-4contribute pre-membrane and envelope proteins.
 34. The method of claim25, wherein the serotype, dengue-4, is present in at least the firstcomposition at a concentration about 1.5 times greater than any otherdengue serotype.
 35. The method of claim 34, wherein dengue-4 is presentin at least the first composition and is at least about 10 times greaterthan dengue-2.
 36. The method of claim 25, further comprisingadministering to the subject at least one additional immunogenic agent.37. The method of claim 25, wherein the first immunogenic compositionand the second immunogenic composition are administered to the subjectsubcutaneously.
 38. The method of claim 25, further comprisingadministering at least one additional booster administration of aformulation of a live, attenuated dengue vaccine after administration ofthe compositions of claim
 25. 39. The method of claim 38, wherein the atleast one additional booster administration is administeredsubcutaneously to the subject after administration of the compositionsof claim
 25. 40. The method of claim 25, wherein at least one additionalsubcutaneous administration of the first immunogenic composition or thesecond immunogenic composition is administered to the subject after theinitial administration.
 41. A method for inducing an immune response ina subject against three or more dengue virus serotypes, the methodcomprising: administering a first immunogenic composition of amonovalent live, attenuated dengue virus; administering a secondimmunogenic composition of three or more live, attenuated dengueviruses, the composition comprising at least one non-chimeric live,attenuated dengue-2 virus and at least one dengue-dengue chimera havinga live, attenuated dengue-2 virus backbone; and; wherein the firstimmunogenic composition and the second immunogenic composition areadministered at two or more anatomical locations consecutively on thesame day to the subject; and inducing an immune response in the subjectagainst the three or more dengue virus serotypes. 42-43. (canceled) 44.The method of claim 41, wherein the second immunogenic compositioncomprises at least three dengue virus serotypes at predetermined ratios.45. The method of claim 41, wherein the second immunogenic compositioncomprises a tetravalent formulation of all four dengue virus serotypes.46. The method of claim 41, wherein the two or more anatomical locationscomprise different anatomical locations on different body parts of thesubject.
 47. The method of claim 41, wherein the two or more anatomicallocations comprise different anatomical locations on the same body partof the subject.
 48. The method of claim 41, wherein the at least onedengue-dengue chimera of a live, attenuated dengue virus comprisesnon-structural proteins from at least one dengue virus serotype andpre-membrane and envelope proteins from at least a second dengue virusserotype.
 49. The method of claim 41, wherein the at least onedengue-dengue chimera of a live, attenuated dengue virus is selectedfrom dengue-2/dengue-1, dengue-2/dengue-3, and dengue-2/dengue-4,wherein dengue-2 is the backbone and dengue-1, dengue-3 or dengue-4contribute pre-membrane and envelope proteins.
 50. The method of claim41, wherein the serotype, dengue-4, is present in at least the secondcomposition at a concentration about 1.5 times greater than any otherdengue serotype.
 51. The method of claim 50, wherein dengue-4 is presentin at least the second composition and is at least about 10 timesgreater than dengue-2.
 52. The method of claim 41, further comprisingadministering to the subject at least one additional immunogenic agent.53. The method of claim 41, wherein the first immunogenic compositionand the second immunogenic composition are administered to the subjectsubcutaneously.
 54. The method of claim 41, further comprisingadministering at least one additional booster administration of aformulation of a live, attenuated dengue vaccine after administration ofthe compositions of claim
 1. 55. The method of claim 54, wherein the atleast one additional booster administration is administeredsubcutaneously to the subject after administration of the compositionsof claim
 1. 56. (canceled)
 57. A vaccine kit for carrying out the methodof claim 41, the kit comprising: an immunogenic composition of three ormore live, attenuated dengue viruses, the composition comprising atleast one non-chimeric live, attenuated dengue virus and at least onedengue-dengue chimera of a live, attenuated dengue virus; and animmunogenic composition of one live, attenuated dengue virus, thecomposition comprising one non-chimeric live, attenuated dengue virus orone dengue-dengue chimera of a live, attenuated dengue virus; at leasttwo devices capable of administering the compositions to a subject. 58.The kit of claim 57, wherein the immunogenic compositions comprisedifferent formulations.
 59. The kit of claim 57, further comprising atleast one immunogenic agent.
 60. The kit of claim 57, wherein theimmunogenic composition of three or more live, attenuated dengue virusescomprises a tetravalent composition of all four dengue virus serotypes.61. The method of claim 25, wherein the first immunogenic compositionand the second immunogenic composition comprise the same composition.62. The method of claim 41, wherein the monovalent live, attenuateddengue virus comprises dengue serotype 4.